1H and 31P Fourier transform magnetic resonance studies of the conformation of enzyme-bound propionyl coenzyme A on the transcarboxylase. The relaxation rates of the carbon-bound protons and of the three assigned phosphorus resonances of propionyl-CoA were measured in solutions of free propionyl-CoA and of the transcarboxylase-propionyl-CoA complex. In free propionyl-CoA, analysis of the 1/T1 values of 15 protons at 100 and 220 MHz and of 1/T1 and 1/T2 of the three phosphorus atoms at 40.5 MHz indicated free rotation of the propionyl region (taur approximately 3 x 10(-11) sec) but hindered motion of the remainder of the molecule with correlation times of 1-3. 5 x 10(-10) sec, approaching the tumbling time of the entire molecule (taur - 6 x 10(-10) sec. The correlation times of the three phosphorus atoms were indistinguishable from those of their nearest neighbor protons. The effects of three homogeneous enzyme preparations with varying contents of Zn(II), Co(II), and Cu(II) on 1/T1 of 12 protons and 3 phosphorus atoms of prionyl-CoA were analyzed with the help of simultaneous equations to yield the individual contributions at the three metal sites. Only diamagnetic effects were detected on the relaxation rates of the three phosphorus atoms. From the diamagnetic effects it was calculated that the motions of the prionyl side chain and of the terminal pantetheine methylene protons were hindered on the enzyme by an order of magnitude (taur approximately 6 x 10(-10) sec) and that the phosphorus atoms were hindered by two orders of magnitude (taur approximately 1 x 10(-8) sec) over the taur values found in free propionyl-CoA, but that these taur values remained well below that of the entire protein molecule (taur =6 x 10(-7) sec)...


Carboxypeptidase inhibitor from potatoes. The effects of chemical modifications on inhibitory activity. The carboxypeptidase inhibitor from Russet Burbank potatoes was subjected to a variety of chemical modifications and their effects on inhibitory activity toward carboxypeptidases A and B were determined. The importance of the alpha carboxylate of glycine-39 to the enzyme-inhibitor interaction was demonstrated by the observation that a derivative in which all four carboxyls were modified was inactive whereas a derivative in which only the beta carboxylates of aspartic acid residues 5, 16, and 17 were masked retained full inhibitory activity. In addition to these three aspartic acid residues, lysine residues 10 and 13, histidine residues 3 and 15, and arginine-32 were modified and residues 1-5 removed with little effect on inhibitory activity. Tryptophan residues 22 and 28 did not react with 2-hydroxy-5-nitrobenzyl bromide or o-nitrophenylsulfenyl chloride, and thus are presumed to be buried in the interior of the inhibitor molecule. Although tyrosine-37 was acetylated without affecting binding characteristics, both carboxypeptidases A and B protected against deacetylation by hydroxylamine. These studies indicate that the carboxyl terminal region of the inhibitor is in contact with enzyme in the complex. The parallel effects of modifications on inhibitory activity toward carboxypeptidases A and B support previous evidence that both enzymes utilize the same binding site on the inhibitor [C. A. Ryan (1971), Biochem. Biophys. Res. Commun. 44, 1265].


Subunit interactions in the methionyl-tRNA synthetase of Bacillus stearothermophilus. The methionyl-tRNA synthetase from Bacillus stearothermophilus is shown to be a dimer of 2 x 82,000 with identical subunits. It exhibits negative cooperativity in substrate binding and "virtual" halt-of-the-sites reactivity. The enzyme binds only 1 mol of methionine in the absence of other ligands, but several methods show that 2 mol of methionyl adenylate are bound per enzyme dimer. However, one of these adenylates is formed 480 times faster than the other (k1 = 29 sec-1 and k2 = 0.06 sec-1). While the rapid phase of the reaction follows normal saturation kinetics with respect to substrate concentration, the rate of the slow phase is independent of substrate concentrations down to 1 muM. It is suggested that the very slow rate of formation of the second adenylate reflects a rate limiting conformational change which precedes a more rapid chemical step on the second subunit.


Interaction of a 5-trans-vinylcarboxylic acid analogue of pyridoxal 5'-phosphate with apoaspartate aminotransferase. Covalent labeling of the enzyme. The 5-trans-vinylcarboxylic acid analogue of pyridoxal 5'-phosphate has been prepared. Its pKa values were determined as 3.08, 4.10, and 7.33. The third pKa, that of the pyridinium nitrogen, is considerably lower than that of 8.2 observed for the corresponding saturated compound, 5'-carboxymethyl-5'-deoxypyridoxal. Absorption spectra of individual ionic forms have been resolved into component bands using lognormal distribution curves. The vinylcarboxylic acid analogue inactivates apoaspartate aminotransferase slowly at pH 8.3. An initial product absorbs at 26 kK (385 nm) and is converted slowly to a species with a narrow absorption band at 24.0 kK (417 nm). Meanwhile, the circular dichroism in the same region changes from positive to negative. At pH 5.2 the product abosrbs at 25.2 kK (397 nm). The 24.0-kK (417 nm) form is not reducible with sodium borohydride and the tightly bound chromophore is not released from the protein during denaturation by acid, base, or heat. L-Glutamate and erythro-beta-hydroxyaspartate both facilitate the formation of the 24.0-kK form. The reaction of the analogue with apoenzyme in the presence of erythro-beta-hydroxyaspartate is also accompanied by transient peaks, presumably representing quinonoid forms, at 19.0 kK (526 nm) and 20.3 kK (492 nm). The analogue reacts at basic pH with arginine, alpha-amino-gamma-guanidinobutyric acid, ornithine, cysteine, alpha, gamma-diaminobutyric acid, eh narrow absorption bands centered in the 24.0-24.4-kK (417-410 nm) region and resembling the product formed with the apoenzyme. Nuclear magnetic resonance and absorption spectroscopy indicate that the reaction with alpha- gamma-diaminobutyric acid proceeds via a hexahydropyrimidine derivative to a substituted tetrahydropyrimidine (a cyclic Schiff base) which is the final product. A similar reaction sequence with the apoenzyme is postulated and a structure with an unknown X group from the enzyme replacing the gamma-amino group of alpha, gamma-diaminobutyric acid is proposed for the 24.0-kK (417 nm) chromophore obtained with the apoenzyme. The proposed reactions are closely related to enzymatic and nonenzymatic reactions of pyridoxal 5'-sulfate (Yang, I. -Y., Khomutov, R. M., and Metzler, D. E. (1974), Biochemistry 13, 3877).


Circular dichroism-inhibitor titrations of arsanilazotyrosine-248 carboxypeptidase A. Coupling of carboxypeptidase with diazotized arsanilic acid specifically modifies a single tyrosyl residue. Yet, owing to the fact that the resultant azoTyr-248 can form an intramolecular chelate with zinc, two different circular dichroism probes result: azoTyr-248 itself and the azoTyr-248-Zn chelate. Both are environmentally sensitive and, characteristically, each can signal the same or different perturbations, as is apparent from circular dichroic spectra. This dual probe function greatly magnifies the scope of these chromophores in mapping the topography of the active center with respect to sites of interaction of inhibitors (or substrates). Titration of the azoenzyme with a series of synthetic, competitive inhibitors, e.g., L-benzylsuccinate, L-phenyllactate, and L-Phe, and with the pseudosubstrate, Gly-L-Tyr, in turn generates characteristic circular dichroic spectra. Their analysis yields a single binding constant for each of these agents, one molecule of each binding to the active center. Mixed inhibitions, as seen with beta-phenylpropionate and phenylacetate, resolved previously into competitive and noncompetitive components, are characterized by different spectral effects. Two molecules of these agents bind to the enzyme, consistent with both thermodynamic and enzymatic studies. The interactions leading to competitive and noncompetitive inhibition, respectively, can be recognized and assigned, based on the manner in which the extrema at 340 and 420 nm, reflecting azoTyr-248, and the negative 510-nm circular dichroism band, typical of its chelate with zinc, are affected and on the pH dependence of spectral and kinetic data. Certai4 noncompetitive inhibitors and modifiers induce yet other spectral features. Each probe is very sensitive to changes in its particular active center environment, though both can be relatively insensitive to inhibitors interacting at a distance from the active center.


Phospholipid exchange between bilayer membrane vesicles. The turbidity of lipid vesicles, freshly prepared by sonicating purified dimyristoyllecithin (DML) in dilute KCl solutions, was measured as a function of time at various temperatures. A sharp maximum in the rate of increase of turbidity is found just above the crystal:liquid-crystal phase transition temperature (Tm). The initial rate of turbidity increase is first order with respect to DML concentration. Electron and light microscopy reveal large vesicles which are not present before incubation or after incubation at temperatures far from the Tm. When temperature, rather than time, is the independent variable, a sharp drop in turbidity is seen at the Tm. The magnitude of this drop and the temperature at which it occurs were used to measure the rate of lipid transfer between vesicles composed of different lipids. A mixture of DML vesicles and dipalmitoyllecithin (DPL) vesicles exhibits sharp drops in turbidity at 24 and 41 degrees, the corresponding Tm's. With time, the magnitude of the transition at 24 degrees decreases while that which was originally at 41 degrees moves to lower temperatures and increases in magnitude. At equilibrium there is a single transition at 32.5 degrees characteristic of vesicles composed of equimolar DPL and DML. The rate at which equilibrium is approached increases at around 24 degrees and again around 41 degrees. These observations indicate that vesicles are in equilibrium with monomolecular lipid, the concentration of the latter being higher the shorter the lipid acyl group or the smaller the vesicle. DML molecules are therefore lost from small vesicles to large vesicles (DML system) or lost from DML vesicles to DML-DPL vesicles (mixed system). When DML vesicles containing a few percent brain gangliosides were studied, different behavior was observed; the initial rate of increase of turbidity becomes second order in lipid concentration, and the rate constant increases with increasing concentrations of KCl. The kinetic order, coupled with the fact that electrolyte reduces intervesicle electrostatic repulsion, argues that in this situation the mechanism of vesicle growth requires vesicle collision.


Phenylalanyl-tRNA synthetases of rat liver: differential effects of thyroid hormone. Thyroxine and analogues inhibit rat liver aminoacyl-tRNA synthetase activity for phenylalanine and tyrosine. A high yield purification of the major cytoplasmic form of phenylalanyl-tRNA synthetase (C1) and its characterization is reported. Polyribosome-bound and other sedimentable forms are found to be indistinguishable from soluble enzyme by immunoprecipitation. Mitochondrial phenylalanyl-tRNA synthetase (M) and cytoplasmic activity (C2) resistant to anti-C1 antibody have been partially purified and characterized. Tissue levels of the three forms are estimated at 22, 1.8, and 4.1 units/g of liver for C1, C2, and M, respectively [1 unit = 1 nmol of Phe-tRNA/min, 30 degrees C]. Charging capability toward rat liver and yeast tRNA, kinetic parameters, and physical properties are compared. Only enzyme C1 is hormone inhibited [K1 = 4 x 10(-6) M for triiodothyronine]. The data indicata that C2 and M are not structurally related to C1; C2 may represent an independent cytoplasmic pool of M. Implications of C1 inhibition in relation to effects on liver protein synthesis are discussed.


The mechanism of quinonediimine acceptor activity in photosynthetic electron transport. The rates of electron flow catalyzed by a variety of unsubstituted and C- or N-methylated quinonediimine electron acceptors in a reaction requiring photosystem II in KCN-inhibited chloroplasts vary according to the structure of acceptor used. Quinonediimine, but not quinone, electron acceptor activities are inhibited by a variety of uncouplers. Kinetic analysis of this inhibition shows that it is competitive. Low concentrations of aniline also inhibit the activity of C-methylated quinonediimines, but this appears to be due to a chemical reaction between the acceptor and aniline at low pH inside the chloroplast. Light-induced uptake of a quinonediimine, p-phenylenediimine, was shown to occur in a DCMU-sensitive reaction. Methylamine uncoupling inhibits this uptake to the same extent as it inhibits electron flow. Experiments with a lipophobic acceptor, N,N,N',N'-tetramethyl-p-phenylenediimine, indicate that it catalyzes electron flow by the same mechanism as other quinonediimines. A model is proposed to account for quinonediimine-catalyzed electron flow.


Characterization of human, bovine, and horse antithrombin III. A comparison of the physical-chemical properties of human, bovine, and horse antithrombin III has been made. These three plasma proteins are strong inhibitors of bovine factor Xa and form a 1:1 molar complex with this coagulation enzyme. Human, bovine, and horse antithrombin III are glycoproteins containing hexose, hexosamine, and neuraminic acid. The total carbohydrate was 9, 12, and 16% for human, bovine, and horse antithrombin III, respectively. These proteins have a similar amino acid composition, although some monor variations were noted. Each antithrombin III is composed of a single polypeptide chain with an amino-terminal histidine residue. Of the first 17 amino-terminal residues, only three differences were noted between the three proteins. These occur in position 2 which is occupied by Gly, Arg, and Trp in human, bovine, and horse, respectively; position 6 which has a deletion in human antithrombin III; and position 8 where Ile in human and horse antithrombin III has been replaced by Val in the bovine preparation. The remainder of the first 17 residues is the same in all three proteins. The molecular weights for the bovine and horse preparation were 56 600 and 52 500, respectively, as determined by sedimentation equilibrium in the presence of guanidine hydrochloride. Some immunological cross-reactivity was also observed between the three different proteins.


Inhibition of bovine factor IXa and factor Xabeta by antithrombin III. Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.


Influence of globin structures on the state of the heme. Ferrous low spin derivatives. Studies of high spin ferrous and ferric derivatives led us to conclude that in the quaternary R structure the state of the hemes is similar to that in the free alpha and beta subunits, but in the T structure a tension acts on the hemes which tries to pull the iron and the proximal histidine further from the plane of the porphyrin. We have now studied the effect of inositol hexaphosphate (IHP) on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO. IHP failed to switch the quaternary structure of carbonmonoxy- and oxyhemoglobin A to the T state, but merely caused a transition to an as yet undefined modification of the R structure. IHP is known to cause a switch to the T structure in hemoglobin Kansas. We have found that this switch induces red shifts of the visible alpha and beta absorption bands and the appearance of a shoulder on the red side of the alpha band; these changes are very weak in carbonmonoxy- and slightly stronger in oxyhemoglobin Kansas. As already noted by previous authors, addition of IHP to nitrosylhemoglobin A induces all the changes in uv absorption and CD spectra, sulfhydryl reactivities, and exchangeable proton resonances normally associated with the R leads to T transition, and is accompanied by large changes in the Soret and visible absorption bands. Experiments with nitrosyl hybrids show that these changes in absorption are caused predominantly by the hemes in the alpha subunits. In the accompanying paper Maxwell and Caughey (J. C. Maxwell and W. S. Caughey (1976), Biochemistry, following paper in this issue) report that the NO in nitrosylhemoglobin without IHP gives a single ir stretching frequency characteristic for six-coordinated nitrosyl hemes; addition of IHP causes the appearance of a second ir band, of intensity equal to that of the first, which is characteristic for five-coordinated nitrosyl hemes. Taken together, these results show that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes, biased toward five-coordinated hemes in the alpha and six-coordinated ones in the beta subunits. The reason why IHP can switch nitrosyl-, but not carbonmonoxy- or oxyhemoglobin A, from the R to the T structure is to be found in the weakening of the iron-histidine bond by the unpaired NO electron and by the very short Fe-NO bond length.


An infrared study of NO bonding to heme B and hemoglobin A. Evidence for inositol hexaphosphate induced cleavage of proximal histidine to iron bonds. Five- and six-coordinate nitrosyl hemes have been prepared and their infrared, electron paramagnetic resonance (EPR), and visible-Soret spectra compared with the corresponding spectra for nitrosyl hemoglobin A (Hba-NO) determined both in the presence and the absence of inositol hexaphosphate (IHP).  The five- and six-coordinate NO complexes prepared from either dipyridine or pyridine carbonyl protoheme dimethyl ester had N-O stretch bands (nuno) near 1675 and 1625 cm-1, respectively.  These frequencies are sensitive to change in solvent (nuno decreased as the dipole moment of the solvent increased) and, with six-coordinate species, to changes in trans ligand.  However, these solvent and trans ligand effects were small compared with the difference (ca. 50 cm-11) between five- and six -coordinate species.  The nature of the trans ligand affected the relative proportions of the two...


Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells. Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.


Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength. Proteins were extracted from isolated rat liver nuclei with 0.15 M NaCl and 0.35 M NaCl at pH 8.0. The number of phosphoproteins in these extracts was determined by labeling with 32P and autoradiography after two-dimensional gel electrophoresis. Two proteins, B22p and B24p, contained small amounts of 32P and sedimented with the 30S nuclear informofer particle. With the exception of two phosphoproteins, CB and CN', all of the phosphoproteins found in the 0.35 M NaCl extract. Approximately 20% of the 0.15 M NaCl soluble proteins bound to rat liver DNA in 0.05 M KCl-0.05 M Tris-HCl (pH 8). Of these proteins, 1-2% bound to DNA in 0.15 M KCl and were eluted with 2 M KCl. This DNA bound fraction which contained both phosphorylated and nonphosphorylated proteins was similar in both the 0.15 and 0.35 M NaCl extracts. However, two major proteins (C13 and C14) and three minor proteins (C15, C25, Cg') were present only in the 0.15 M NaCl extract. The results of the present study show that there are marked similarities in the two-dimensional gel electrophoretic, phosphorylation, and DNA binding properties of rat liver nuclear proteins soluble in either 0.15 or 0.35 M NaCl.


Circular dichroism studies on the alpha-D-galactopyranosyl binding lectin isolated from the seeds of Bandeiraea simplicifolia. The conformation of the alpha-D-galactopyranosyl binding lectin isolated from Bandeiraea simplicifolia seeds has been investigated over a broad range of pH in the presence of various solvents by circular dichroism (CD) spectroscopy in the region 200-300 nm. Analyses of the spectra obtained on the native protein show the lectin to contain a considerable proportion of beta structure (30-40%). The native conformation was found to be largely insensitive to changes in pH, but was influenced by sodium dodecyl sulfate or trifluoroethanol. Alterations in conformation in the presence of these agents were reflected in the CD spectra and show the presence of alpha helix under these conditions. These changes in conformation are accompanied by a loss in polysaccharide-precipitating activity. The protein is irreversibly denatured in 8 M urea. Neither removal of the intrinsic calcium ions from the protein nor addition of methyl alpha-D-galactopyranoside induces any appreciable change in the CD spectra of the protein although the former treatment abolishes the polysaccharide-precipitating capacity of the lectin. The conformational data obtained in the present study are compared with data available from conformational studies of other lectins and leads to the hypothesis that most lectins probably contain beta structure as the predominant conformational feature.


Separation of chromatin containing bromodeoxyuridine in one or both strands of the DNA. A method has been devised for the separation of chromatin containing 5-bromodeoxyuridine (BrUdRib) in one strand (HL) of the DNA from that with BrUdRib in both strands (HH). Ultraviolet light breaks chromatin containing HH DNA into smaller fragments than chromatin containing HL DNA and the two species can be partially resolved on neutral sucrose gradients. Unfiltered ultraviolet light is not suitable since it causes considerable alteration in the electrophoretic pattern of chromatin-associated proteins. Irradiation with 313-nm light causes much less damage to the associated proteins. The ability to separate, isolate, and examine chromatin containing HL and HH DNA makes studies on the distribution of chromatin-associated proteins possible.


Introduction of purified hexosaminidase A into Tay-Sachs leukocytes by means of immunoglobulin-coated liposomes. To determine whether ligand-receptor interactions could engender the selective uptake by deficient cells of enzyme-laden liposomes, aggregated human IgG was used to coat liposomes which had previously trapped purified hexosaminidase A (Hex A). By a new, high-yield procedure, Hex A was purified 7000-fold from human placenta: the homogeneous protein had a pI of 5.4, permitting nonelectrostatic trapping in the aqueous interstices of anionic multilamellar liposomes (molar ratios of phosphatidyl-choline-dicetyl phosphate-cholesterol, 7:2:1). Trapped Hex A was separated from free enzyme by means of Sephadex G-200 chromatography: 1.3 +/- 0.3 mUnits of Hex A/mumol of phospholipid became associated with liposomes and trapped glucose, utilized as a marker of the aqueous compartment. Once sequestered, the enzyme remained latent until lamellae were disrupted by Triton X-100. Presence of enzyme in aqueous compartments was proved by the demonstration of increased trapping (0.02-1.33 mUnits/mumol of phospholipid) with increments in like-sign repulsion of the bilayers produced by increasing molar ratios of anionic dicetyl phosphate (5-20%). To provide for ligand-receptor interaction with surface Fc receptors of human polymorphonuclear leukocytes (PMN's), liposomes were coated by heat-aggregated (62 degrees C, 10 min) human IgG. PMN's from Tay-Sachs patients genetically deficient in Hex A activity readily incorporated exogenous Hex A provided in this fashion. PMN's exposed to enzyme-laden liposomes coated with aggregated IgG incorporated significantly more Hex A than when the enzyme was presented in uncoated liposomes or in liposomes coated with native IgG, which engages Fc receptors with less avidity. Free enzyme was not endocytized. Acquisition of specific Hex A isozyme activity by cells (determined by DEAE-cellulose chromatography) was not due to surface adsorption since cytochalasin B, which prevents phagocytosis but not surface adherence; blocked uptake. Incorporation of the isozyme by deficient cells was also demonstrated by starch gel electrophoresis, and ultrastructural studies showed that the immunoglobulin-coated, Hex A-containing liposomes were taken up into PMN lysosomes after membrane fusion. The studies indicate that liposomes coated with surface ligands may be used to introduce enzyme or other materials into deficient cells possessing appropriate surface receptors.


High resolution thermal denaturation of DNA: thermalites of bacteriophage DNA. High resolution thermal denaturation profiles are presented for the DNAs of bacteriophages lambda and T7. It is concluded that the temperature increment in data gathering and the method of calculating results meet the requirements for quantitative recording of the large amount of information found in the thermal transitions of both DNAs. The high resolution derivative denaturation profiles of these bacteriophage DNAs demonstrate that individual subtransitions (thermalites) of natural DNA are Gaussian in form and have narrow transition widths. Curve resolution performed on these profiles indicates that the mean thermalite width (2 sigma) is 0.33 degrees C and that this breadth is relatively invariant. Transition widths are not influenced by the position of thermalites in the profile or by cation concentration in the range from 5 to 30 mM Na+. However, the relative position of thermalites within a denaturation profile is a function of the solution ionic strength. The distribution of lengths of the DNA sequences which these thermalites represent is broad, with a number average length of 900 base pairs. Although we find an approximate similarity between the number of thermalites in the denaturation profile of T7 DNA and the number of looping regions in the electron microscopic partial denaturation map of Gomez and Lang ((1972), J. Mol. Biol. 70, 239-251) we conclude that free solution thermal denaturation experiments can be compared only superficially to the mapping results.


Conformational properties of adenylyl-3' leads to 5'-adenosine in aqueous solution. A detailed 220-MHz NMR study has been made of the conformational properties for the homodinucleotide adenylyl-3' leads to 5'-adenosine, ApA, in D2O. Unambiguous signal assignments of all proton signals were made with the aid of selectively deuterated nucleotidyl units, ApA, ApA, and D-8ApA, and complete, accurate sets of NMR parameters were derived by simulation-iteration methods. Sets of limiting chemical shifts and coupling values were also obtained for ApA and constituent monomers 3'-AMP and 5'-AMP at infinite dilution and at identical ionization states for assessment of dimerization effects. Conformational properties were evaluated quantitatively for most of the conformational bonds of ApA and these are consistent with two compact folded dynamically averaged structures, a base-stacked right helical structure, I, characterized as anti, C3'-endo, g-, w,w' (320,330 degrees), g'g', gg, C3'-endo, anti, and a more loosely base-stacked loop structure, II, with anti, C3'-endo, g-, w,w' (80 degrees, 50 degrees), g'g', gg, C3'-endo, anti orientations. Dimerization produces a number of nucleotidyl conformational changes including a shift in ribose equilibrium C2'-endo (S) in equilibrium C3'-endo (N) in favor of C3'-endo in both Ap- and -pA (60:40 vs. 35:65 in monomers), a change in glycosidic torsion angle chiCN toward 0 degrees, and a greater locking-in of rotamers along bonds involved in the phosphodiester backbone. Moreover, there is clear evidence that the transitions from S leads to N forms and chiCN leads to 0 degrees are directly related to base stacking in ApA. Finally, ApA exists in solution as an equilibrium between I, II and an unstacked form(s) with as yet undetermined conformational features. Since C4'-C5', C5'-O5', and C3'-O3' bonds possess exceptional conformational stabilities, it is proposed that destacking occurs primarily by rotation about P-O5' and/or O3'-P. Predominant factors influencing the overall ApA conformation are thus base-base interaction and flexibility about P-O5' and O3'-P, with change of ribose conformation occurring in consequence of an alteration of chiCN, the latter in turn being governed by the need for maximum eta overlap of stacked adenine rings.


Conformational studies of peptide cyclo-(D-Val-L-Pro-L-Val-D-Pro]3, a cation-binding analogue of valinomycin. The solution conformation of cyclo-[D-Val-L-Pro-L-Val-D-Pro]3 (PV) and its alkali-metal ion complexes was investigated by proton nuclear magnetic resonance spectroscopy. It is concluded that the cation complexes of PV have S6 symmetry and are essentially isostructural with the K complex of valinomycin. In contrast to valinomycin, the Li- and Na-PV complexes are stable in methanol and have dissociation rate constants that are several orders of magnitude slower than the corresponding valinomycin complexes. Also in contrast to valinomycin, free PV exists in two different conformational states which interconvert at very slow rates (less than 1 s-1). One of these conformers has S6 symmetry and is structurally similar to that of the cation complexes. The other species, which has lower symmetry than S6, is the more stable conformer. Depending upon concentration and solvent polarity, the latter represents between 50 and 75% of the total mixture. It is proposed that PV may have a higher affinity for cations than valinomycin because of its higher potential energy in the uncomplexed state.


Photoreceptor protein from the purple membrane of Halobacterium halobium. Molecular weight and retinal binding site. The apparent molecular weight of the purple membrane protein of Halobacterium halobium was found to be 20 000 by sodium dodecyl sulfate gel electrophoresis and by gel filtration in sodium dodecyl sulfate. However, the molecular weight value determined by gel filtration in 6 M guanidine was 28 000. To resolve this discrepancy, methods insensitive to or independent of the conformation of the protein were used to estimate the molecular weight. Analytical ultracentrifugation of the sodium dodecyl sulfate-protein complex, peptide mapping, and amino acid analysis all gave values of 25 000 +/- 1000, a figure in agreement with a recent x-ray study. Borohydride reduction was used to attach the retinal cofactor covalently to a lysine residue. After digestion with thermolysin, peptide maps were prepared of the protein labeled at lysine residues with [14C] succinic anhydride both before and after reduction. Comparison of the maps showed one radioactive peptide with changed mobility. This peptide was isolated and shown to have the sequence Val-Ser-Asp-Pro-Asp-Lys-Lys with only one of the two lysine residues alkylated. Solid-phase sequencing showed the succinyl group to be at position 6 and hence the retinal group to be at position 7. It was possible that a small amount of retinal was also bound to Lys-6. There was no apparent homology with the corresponding peptide of vertebrate rhodopsin. No evidence of chain heterogeneity was found by radiochemical peptide mapping and sequence analysis of peptides containing lysine residues indicating that all protein chains of purple membrane are very similar or identical.


Analysis of the two steps in polypeptide chain initiation inhibited by pactamycin. Earlier work has shown that the inhibition by pactamycin (PM) of polypeptide chain initiation in reticulocyte extracts is associated with (1) a defect in the joining of the 60S subunit to the smaller initiation complex to form an 80S complex ("joining reaction") (Kappen, L. S., Suzuki, H., and Goldberg, I. H. (1973), Proc. Natl. Acad. Sci. U.S.A. 70, 22) and (2) a block after the synthesis of the initial dipeptide (Kappen, L. S., and Goldberg, I. H. (1973), Biochem. Biophys. Res. Commun. 54, 1083). The relative contributions of these two effects to the action of PM and their relationship to one another were evaluated in a system employing sparsomycin that permits both initiation at a certain number of initiation sites and limited oligopeptide formation without termination and release. The degree to which PM blocks the "joining reaction" and leads to the accumulation of 48S initiation complexes that either remain free or are bound to polysomes without the corresponding 60S subunit ("half-mers") was estimated by treatment of polysomes with RNase. Met-tRNAfMet binding factors are required to stabilize the RNase-generated 48S complexes. Under conditions where the initiation factor required for the "joining reaction" functions catalytically, presumably by cycling on and off initiation complexes, PM usually inhibits 80S complex formation 50-70%. Where "joining" is not limiting (presence of at least stoichiometric amounts of joining factor or high Mg2+ concentration) PM leads to the maximal accumulation of the initial dipeptide, Met-Val, in the P-site on the ribosome, indicating a block in a subsequent step in elongation. Binding studies with [3H]PM and the inability of PM to inhibit elongation of preformed Met-Val indicate that PM must interact with the ribosomes at an early stage of initiation. Taken together these data are compatible with the suggestion that PM does not interfere with the ribosomal "joining reaction" per se, but prevents the release and reuse of the joining factor, and in so doing blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome.


RNA synthesis in isolated hen oviduct nuclei. Nuclei have been prepared from the oviduct of the adult laying hen which are capable of synthesizing large amounts of RNA for long periods of time. The time course of RNA synthesis is linear through 3 h of incubation after an initial burst of activity and is inhibited 60-70% by alpha-amanitin. Maximum synthetic activity requires the presence of serum albumin to stabilize the nuclei, high concentrations of the four ribonucleoside triphosphates, and an incubation temperature of 25 degrees C for continued linear synthesis beyond 30 min. The RNA synthesized in vitro is predominantly 10-20 S with a small proportion of higher molecular weight product. Much of the 10-20S RNA is probably transcribed by RNA polymerase II and is of a size comparable to ovalbumin mRNA. A fraction of this RNA appears to contain poly(A) sequences suggesting that there is some processing of the newly synthesized RNA. These nuclei may provide a useful system for studying the control of the transcription and maturation of ovalbumin mRNA in vitro.


Rat liver cytoplasmic glucose-6-phosphate dehydrogenase. Steady-state kinetic properties and circular dichroism. Steady-state kinetic studies including initial velocity, NADPH product inhibition, dead-end inhibition, and combined dead-end and product inhibition measurements with purified rat liver glucose-6-phosphate dehydrogenase indicate a sequential and obligatory addition of substrates in the order of NADP+, glucose-6-P for the catalytic pathway at pH 8.0. Although instability of 6-phosphoglucono-delta-lactone precluded product inhibition experiments which might directly exclude an enzyme-6-phosphoglucono-delta-lactone complex, the absence of an enzyme-glucose-6-P complex suggests that the enzyme-lactone product is unlikely and the release of products is also ordered, with NADPH released last. Consideration of the kinetic constants (Ka = 2.0 muM, Kiq = 13 muM) and cellular concentration of the substrates and products suggests extensive inhibition of the enzyme in vivo and control by the NADPH/NADP+ ratios. Circular dichroism spectra of the enzyme in 20 mM phosphate buffer at pH 7.0 and 25 degrees C indicate 51% helix and 33% pleated sheet structures which is considerably different from results (14% helix) with yeast enzymes.


Lipoprotein lipase: evidence for high- and low-affinity enzyme sites. The kinetic constants for membrane-supported lipoprotein lipase have been determined for the enzyme active in lipoprotein triglyceride catabolism in perfused heart and adipose tissues, using a nonrecirculating system. Heart endothelial lipoprotein lipase reacted as a single population of high-affinity substrate binding sites (Km' 0.07 mM triglyceride). Km' (apparent Michaelis constant for the supported enzyme species) was independent of flow rate and the enzyme was rapidly released by heparin, suggestive of a superficial membrane binding site. Lipoprotein lipase active in perfused adipose tissue had significantly different kinetic properties, including a low substrate affinity (Km' 0.70 mM triglyceride), diffusion dependence of Km' at low flow rates, and slow release of enzyme by heparin. Adipose tissue may contain a small proportion of high affinity sites. While only a small proportion of total heart tissue lipoprotein lipase was directly active in triglyceride hydrolysis, this study suggests that the major part of lipoprotein lipase in adipose tissue may be involved in the hydrolysis of circulating lipoprotein triglyceride.


Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells. X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.


Rapid, preparative-scale purification of chromatin proteins. Methods are desceibed which permit rapid isolation of chromatographically purified histone and non-histone chromatin proteins under relatively mild chemical conditions. Chromatin is isolated from purified nuclei, dissociated in guanidine - HCl-urea and the nucleic acids removed by ultracentrigugation. This can be accomplished in 10 h by employing maximum-force rotors (500 000 x g). The proteins are then fractionated by a batch ion-exchange method, which leads to a rapid and complete separation of the histones and non-histone components, in apparently undegraded form. With these methods it is possible to obtain mg quantities of chromatographically pure histone and non-histone proteins in less than a single working day.


Purification of sea urchin ribosomal RNA genes with a single-strand specific nuclease. Ribosomal RNA genes (rDNA) from Lytechinus variegatus were isolated by selective heat denaturation of main band DNA followed by single-strand specific nuclease (S1) treatment to remove the single-stranded DNA. After S1 nuclease treatment the partially purified fraction contained 10% rDNA, representing a 50-fold purification. Preparative CsCl centrifugation of this fraction resulted in highly purified rDNA with an average molecular weight of 1.9 - 10(7) and no single-strand breaks. High molecular weight sea urchin DNA was refractory to selective heat denaturation. DNA with an average molecular weight of greater than or equal to 2.9 - 10(7) was only 60-80% denatured after heating 13 degrees C above the Tm, whereas, DNA with an average molecular weight of less than or equal to 1.9 - 10(7) was 98% denatured. This phenomenon appears not to be due to time, buffer, or pH, but is dependent on size.


The binding site for coat protein on bacteriophage Qbeta RNA. The site of interaction of phage Qbeta coat protein with Qbeta RNA was determined by ribonuclease T1 degradation of complexes of coat protein and [32P]-RNA obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and bound [32P]RNA fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Qbeta, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.


Regulation of ternary (Met-tRNAf - GTP - eukaryotic initiation factor 2) protein synthesis initiation complex formation by the adenylate energy charge. Formation of the ternary [Met-tRNAf - GTP - eukaryotic initiation factor 2] protein synthesis initiation complex in rabbit reticulocyte ribosomal eluates is dependent on the GTP: GDP ratio and on the adenylate energy charge. The elements controlling ternary initiation complex formation have been studied in a reconstituted system contianing eukaryotic initiation factor 2 and nucleoside diphosphate kinase purified from the ribosomal eluate. The concentration of GTP required for half maximal formation of the ternary initiation complex is 2.5 - 10(6) M; GDP is a potent competitive inhibitor with Ki equals 3.4 - 10(7) M. Sensitive control of ternary initiation complex formation by the adenylate energy charge occurs through nucleoside diphosphate kinase regulation of the GTP : GDP ratio. Over a wide range of GTP : GDP ratios, 50% of maximal ternary initiation complex formation is observed at an adenylate energy charge of 0.85-0.90 resembling that seen in the unfractionated system. Small changes in adenylate energy charge near this value result in significant changes in the extent of ternary initiation complex formation. Since GTP is continually hydrolyzed to GDP during protein synthesis and since GDP is a competitive inhibitor of GTP binding to several of the protein factors necessary for mRNA translation, the synthetic process provides sensitive control by product inhibition. Ribosome-associated nucleoside diphosphate kinase control of GTP regeneration in response to the adenylate energy charge provides one mechanism for linking protein synthesis to the nutrient state and energy charge of the cell.


The crystal and molecular structure of an osmium bispyridine adduct of thymine. The bispyridine osmium adduct of thymine has been crystallised and subjected to an X-ray diffraction analysis. It crystallises in the triclinic space group P1, with cell dimensions a equals 7.975(3), b equals 10.381(3), c equals 11.036(3) A, alpha equals 82.73(2)degrees, beta equals 77.22(3) degrees, gamma equals 101.75(3), and with two molecules in the unit cell. The analysis has shown that the osmium reagent has added cis across the 5,6 thymine bond.


Diacridines: bifunctional intercalators. I. Chemistry, physical chemistry and growth inhibitory properties. The synthesis, as well as the rationale for synthesis of diacridines, double intercalators, as potential inhibitors of nucleic acid synthesis is presented. The syntheses of (9-acridyl)-putrescine and -spermine, and bis(-9-acridyl)-putrescine, -spermidine, -spermine diamines and of bis(6-chloro-2-methoxy-9-acridyl)-putrescine and -spermine diamines, all substituted on the terminal NH2 groups are described. In addition, the homologous series of diacridines connected by the amino groups of the diamines NH2(CH2)nNH2 (where n = 2,3,4,6,8,10,12,14,16,18) to the C-9 of the diacridines has been synthesized. The chemical properties of these compounds as well as their molecular relationship to DNA are presented. The effect of the double intercalators on the Tm of DNA and of (A)n - (U)n, (dA)n - (dT)n, (G)n - (C)n and on (dG)n - (dC)n have been determined. The double acridine intercalators produce a much greater increase of the Tm of these nucleic acids than do the single acridine intercalators. They also profoundly affect the Tm of DNA in physiological salt concentrations; under these latter conditions the single intercalators have no effect. The relationship between the length of the chain connecting the two acridine rings and the inhibition of the growth of P-388 cells in vitro and vivo is presented. Their growth inhibitory properties appear, in general, to parallel their intercalative abilities.


Diacridines: bifunctional intercalators. II. The biological effects of putrescine, sperimidine and spermine diacridines on HeLa cells and on the L-1210 and P-388 leukemia cells. The effect of putrescine, spermidine and spermine diacridines on the growth of HeLa cells and of P-388 and L-1210 leukemia cells has been evaluated and compared to that of the parent compound, 9-aminoacridine. The diacridines are more effective growth inhibitors than 9-aminoacridine. The primary site of action appears to be the inhibition of RNA synthesis.


Diacridines: bifunctional intercalators. III. Definition of the general site of action. Electron microscopy of HeLa cells exposed to spermine diacridine shows nucleolar distortions which disappear after several days despite the persistence of the metabolic changes promoted by spermine diacridine. This compound inhibits ribosomal RNA synthesis and appears to act independently of any particular phase of the cell cycle. The DNA content of the HeLa cells remains unchanged and the cell distribution is not significantly disturbed from its normal distribution in the various phases of the cell cycle. Spermine diacridine and other diacridines inhibit primarily chain initiation but also chain elongation by DNA-directed RNA polymerase of Azotobacter vinelandii.


DNA synthesis and repair in permeable cells of Micrococcus radiodurans. Cells permeable to deoxyribonucleoside triphosphate were prepared from Micrococcus radiodurans, and DNA synthesis and rejoining of strand scissions induced by gamma-rays were investigated. DNA synthesis was stimulated by ATP at an optimal concentration of 1mM. This reaction requires four deoxyribonucleoside triphosphates and MgCl2. NAD inhibited the reaction, but no rejoining of primer DNA was observed. Even in the presence of NAD, DNA which was synthesized in the unirradiated permeable cells had a peak molecular weight of only 1.3 - 10(6). DNA synthesis was stimulated by irradiation of the permeable cells with gamma-rays, but this stimulatory effect was eliminated by the addition of NAD. Both primer and synthesized DNA in the irradiated permeable cells were rejoined in vitro in the presence of NAD and deoxyribonucleoside triphosphates, while those in the unirradiated permeable cells were not rejoined.


Purification and characterization of RNA polymerase I from a higher plant. RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density  gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).


Ribosomal RNA gene dosage as a function of tissue and age for mouse and human. The average number of rRNA genes per haploid genome (rRNA gene dosage) of the cells present in liver and brain was determined throughout the lifespan of the inbred C57BL/6J mouse strain and of human. Ribosomal RNA gene dosage was determined using the RNA-excess DNA - RNA hybridization technique. DNA was extracted and purified using a CsCl/chloroform method with a high percent yield (over 90%) to minimize any possible effects of tissue and age-dependent selective loss or gain of rRNA genes. Radioactive rRNA was from the liver of the youngest age group for either mouse or human in all hybridization experiments, with DNA from the different tissues and age groups being the only variable. In the young mouse (35-49 days), the rRNA gene dosage was 36% higher in brain (114 genes), as compared to liver (84 genes). The rRNA gene dosage remained  essentially constant as a function of age for mouse brain; but between the age of about 220 to 440 days, it increased in liver, attaining approximately an equal value to that of brain. No significant difference was found in the rRNA gene dosage of brain or liver between different mice of the same age. In contrast to this result, a significant difference was found between human tissues of similar age. The rRNA gene dosage ranged about 2-fold (148-289) between 2 months to 75 years of age. An age-dependent trend, similar to that for mouse liver, was found when the averages of four different age groups totaling 20 individuals were compared. However, this was not statistically significant. No difference in the rRNA gene dosage as a function of sex or tissue was apparent. Several models are discussed to account for these results.


Inhibition of protein and nucleic acid synthesis of animal cells in vitro mediated by high molecular weight inhibitors in human liver extract. 1. The addition of human liver extract to HeLa cells induces a reversible inhibition of the incorporation of [3H] thymidine into the DNA, [3H] uridine into the RNA, and 14C-labelled amino acids into the protein of HeLa cells. The inhibitory effects appear after treatment for 1 h and reach a maximum after 4-8 h. These effects do not depend on a defective precursor penetration, isotopic dilution or degradation of labelled precursor (thymidine-degrading enzymes were inactivated by the addition of unlabelled thymine), reduced activity of thymidine and uridine kinase, medium impairment, or an impairment of the cell-membrane function. 2. The nucleic acid synthesis-inhibiting activity of the extract seems to be dependent on cellular protein synthesis but independent of RNA synthesis which indicates that the inhibitors act in an indirect way. Furthermore, the inhibitors seem to lack the tissue-specific character of chalones. 3. The extract contains separate inhibitors of DNA, RNA and protein synthesis. These inhibitors were found to have different physical-chemical characteristics and to be macromolecules with a protein or conjugated protein character (mol. wt. approx. 90 000). 4. The possibility that the activity of the high molecular weight inhibitors resides in low molecular weight factors (bound to protein carriers) was tested: No true low molecular weight inhibitors could be liberated by extraction with trichloroacetic acid/organic solvents or by dialysis/enzymatic treatments. Nucleosides such as thymidine, uridine, and cytidine, however, were liberated and could be shown to interfere with the uptake of [3H] thymidine/[3H] uridine.


On the hydrophobic part of aminopeptidase and maltases which bind the enzyme to the intestinal brush border membrane. The intestinal brush border aminopeptidase and unfractionated maltases M2+M3 are composed of a hydrophilic, sugar containing and enzymatically active part, and a smaller hydrophobic part presumably binding the catalytic part of the lipid matrix of the membrane. Hydrophobic parts detaced by trypsin from the detergent forms of aminopeptidase and the maltases were purified and shown to have molecular weights ranging from 8000 to 10000. All are rich in hydrophobic residues and contain no disulfide bridges. However, their overall amino acid composition is different. The hydrophobic parts appear to be N-terminal in the detergent forms of the enzymes.


Water binding and mobility in the phosphatidylchlone/cholesterol/water lamellar phase. Measurements of hydration and water self diffusion in lamellar phases of the ternary system: phosphatidylcholine/cholesterol/water have been made using pulse NMR relaxation methods. Systems containing phosphatidylcholine and cholesterol in a 1:1 mol ratio with varying water contents are studied at 20.5 degrees C. The results indicate that 12 water molecules corresponds to complete hydration of the phosphatidylcholine/cholesterol unit, and in the region of this hydration a 4-fold decrease in water diffusion occurs. The nature of the bound water and its relationship to phase stability and overall water mobility in the system are discussed. It is concluded that at the stoichiometric composition the diffusion decreases due to the relative immobility of the bound water. The implications in terms of permeability regulation in the aqueous channels by water content and hydration are cited.


Raman spectroscopic investigations of sarcoplasmic reticulum membranes. Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.


Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria. Comparison with microsomes. 1. Mitochondria, inner and outer mitochondrial membranes and microsomes were isolated and purified from pig heart. Their lipid composition and protein components were studied. 2. The fatty acid distribution in the main phospholipids seemed specific rather of a given phospholipid and not of one type of membrane. 3. Inner mitochondrial membranes were characterized by a high content in cardiolipin and a very low level of triglycerides together with a high degree of unsaturation and C18 acids. Gel electrophoresis revealed 13 different polypeptide subunits of which 5 were major ranging in molecular weights from 10000 to 215000. 4. In outer mitochondrial membranes, total lipid, phosphatidylcholine, phosphatidylinositol, plasmologen and triglyceride contents were much higher than in inner membranes. Fatty acids of phospholipids were mostly saturated and the polypeptide pattern showed 12 components, of which 4 were major of mol. wt 75000, 60000, 20000 and below 10000. 5. Compared to outer membrane, microsomes exhibited a much higher cholesterol content and markedly different protein profiles. They contained significant amounts of cardiolipin and phosphatidylserine, this latter phospholipid being exclusively located in microsomes. However odd similarities were observed in some lipid components of microsomes and inner mitochondrial membranes, but fatty acids were more saturated in microsomes and electrophoretic profiles of protein components appeared very different and revealed components of high mol. wt.


Nitrobenzylthionionosine binding sites in the erythrocyte membrane. Nitrobenzylthioinosine binds tightly, but reversibly, to sites in the human erythrocyte membrane; occupancy of these sites blocks the transport of uridine and of other nucleosides. This report described the inhibition of nitrobenzylthioinosine binding at these sites by substrates of the uridine transport mechanism and by compounds related to nitrobenzylthioinosine. For some of these compounds dissociation constants for binding at the nitrobenzylthioinosine sites were determined, assumming competition with nitrobenzylthioinosine. Deoxycytidine, a substrate for the uridine transport mechanism, did not inhibit binding of nitrobenzylthioinosine, suggesting that binding sites for the latter are distinct from nucleoside sites directly involved in transport.


The inhibitor effect of probencid and structural analogues on organic anions and chloride permeabilities in ox erythrocytes. Probenecid inhibits anion movements (organic anions and chloride) in ox erythrocytes. The I50 is 4. 10(-5) M. Structural analogues such as carinamide, p-carboxybenzene sulfonamide and p-carboxy N,N-diethyl benzene sulfonamide, which are drugs of the sulfonamide class, were also found to inhibit anion transport. These results reinforce the previously discussed view based on structural considerations, that sulfonamides act on the red cell membrane as competitors of anion transport. It is possible that probenecid and carinamide act in a similar way in the kidney.


Sodium ion transport in isolated intestinal epithelial cells. II. Comparison of the effect of actively transported sugars on sodium ion efflux in cells isolated from jejunum and ileum. Na+ transport studies in intestinal epithelial cells indicate that enterocytes from different regions of the small intestine differ in their response to actively transported sugars. 1. Compared with sugar-free medium total Na+ efflux rate constants from isolated rat jejunal cells were significantly increased when medium contained actively transported sugars, glucose and galactose, but not when medium contained fructose. 2 In contrast total Na+ efflux rate constants from isolated rat ileal cells did not respond to actively transported sugars, glucose and galactose. 3. Similar results for the effect of actively transported sugars on Na+ ellux were obtained for isolated rabbit jejunal and ileal epithelial cells. 4. Passive Na+ efflux rate constants for isolated jejunal and ileal enterocytes are not significantly different, indicating similiar permeability characteristics.


Influence of membrane lipid fluidity on glucose and uridine facilitated diffusion in human erythrocytes. A central question which must be resolved before acceptable molecular descriptions of facilitated diffusion systems can be provided is the nature of the spatial and functional relationships between the transport proteins and the membrane lipids. In the work reported here, this question was addressed by investigating the dependence of the rates of glucose and uridine facilitated diffusion in human erythrocytes on membrane lipid fluidity. Two approaches were used to alter the lipid fluidity: treatment with ether, an anesthetic, and the exchange of a synthetic 3-ketosteroid, cholest-4-en-3-one, for membrane chloesterol. Both of these treatments result in a significant increase in membrane lipid fluidity, as judged by the increase in the rates of passive diffusion of uridine through cell membranes and of glucose through membrane lipid bilayer vesicles. Ether produces no change in the Km of either transport process, a slight decrease in the V for glucose transport, and no significant change in the V for uridine transport. Replacement of membrane cholesterol by cholest-4-en-3-one reduces the V for glucose transport slightly, without altering the Km, and reduces both the Km and V for uridine transport. The absence of the expected increase in the V of facilitated diffusion with increasing membrane lipid fluidity observed here with human erythrocytes is not consistent with models for the transport process which feature movement of transport proteins which are in direct contact with the bulk lipids of the membrane.


Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis. L-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na+ dependent and presumably coupled to Na+ gradient. Our results indicate that progesterone (10(-6) M) Blocks abruptly, around the germinal vesicle breakdown, the saturable transport of L-leucine. p-Chloromercuribenzoate (10(-4) M) induces maturation and after a short lag of time strongly inhibits L-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.


Molecular transport in thyroid slices.

Molecular transport in thyroid slices. The aim of this work is to describe quantitatively, from a physical point of view, molecular, molecular transport in thyroid slices. The study of the release of [3H]sucrose, [3H]inulin and 131I-labeled albumin leads to the following conclusions: the molecular transport in the slices is not due mainly to free diffusion. Indeed as these molecules are retained in the interfollicular spaces, the transport of matter is a mechanical process due to the agitation of the medium in which the slices are incubated. It depends on the elastic properties of the thyroid tissue, the frequency of the agitator and the thickness of the slice. This transport process can be described by a diffusion equation with an empirical diffusion coefficient; we call it diffusivity. These findings must be taken into account in any in vitro kinetic study of thyroid metabolism and of its regulation by effectors such as thyrotropin. The possibility of interference of such mechanical processes in the interpretation of kinetic tracer studies with tissue slices or fragments should be considered.


Sensitivity of erythrocyte acethylcholinesterase to inhibition by linolenoyl sorbitol. Dependence on a transmembrane potential. Acetylcholinesterase activity of human erythrocytes is known to be inhibited by linolenoyl sorbitol, the inhibition being critically dependent on cell membrane intactness. The extent of enzyme inhibition by the added lipid is correlated with the magnitude of Cl- gradient across the erythrocyte membrane, indicating that enzyme sensitivity is associated with a transmembrane potential. If linolenoyl sorbitol is allowed to interact with the erythrocytes while a Cl- gradient exists, enzyme sensitivity can subsequently be demonstrated not only in the absence of a gradient but even when the cells are lyzed. It is consluded that the transmembrane potential determines the accessibility of a membrane component to the added lipid.


Prostaglandin E1 high affinity binding sites of rat thymocytes. Specificity and blockade by non-steroidal antiinflammatory drugs and localization in a plasma membrane-enriched fraction. Great specificity is demonstrated for the prostaglandin E1 high affinity binding sites of rat thymocytes. Whereas prostaglandin E2 has the same affinity as prostaglandin E1, 13 other prostaglandin derivatives and antagonists are bound with 2-1000 times smaller affinities. 50% inhibition of the high affinity binding of prostaglandin E1 to rat thymocytes is demonstrated for three non-steroidal antiinflammatory drugs, indomethacin (3.6. 10(-5) M), salicylic acid (2.9. 10(-3) M) and acetylsalicylic acid (2.10(-2) M). The low affinity binding of prostaglandin E1 is enhanced by the same concentration of indomethacin, however, to a lesser degree and more variable than the inhibition of the high affinity binding of prostaglandin E1. Like intact cells a 50-fold purified plasma membrane fraction, isolated from a homogenate of rat thymocytes, shows reversible high affinity binding of prostaglandin E1 as well as irreversible binding of unidentified tritiated compounds. The binding  data are compatible with a localization in the plasma membrane of high affinity sites for reversible binding with a considerably higher dissociation constant than that found for whole cells. Their identity remains to be demonstrated.


Phase transitions in 1,2- and 1,3-diacyl-sn-glycero-3-phosphocholine monolayers at the oil/water interface. Moving the phosphatidylcholine group from the 3- to the 2-position in monolayers of distearoyl-sn-glycero-3-phosphocholine at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.


The effects of removal of sodium ions from the mucosal solution on sugar absorption by rabbit ileum. Net absorption and accumulation of D-galactose, beta-methyl D-glucose and low concentrations of 3-0-methyl-D-glucose by sheets of rabbit ileum are observed even when Na+ in the mucosal solution is replaced by choline. This indicates that active sugar transport can occur in the direction opposite to the brush-border Na+ gradient.


Amino acid transport by the helicoidal colon of the new-born pig. The proximal colon of the new-born pig maintains a stable short-circuit current which is partly dependent upon the presence of methionine. This interaction betweeen methionine and short-circuit current shows Michaelis-Menten knetics with a Km of 0.24 mM and a V of 27 muA.cm-2. The net flux of methionine to the serosal surface of proximal colons also shows a hyperbolic relation to the external concentration of methionine (Km 0.38 mM; V 10.4 nmol.cm-2. min-1). The proximal colon concentrates methionine within its epithelium giving a mucosal to medium ratio of 11.2 +/- 1.9 (90 min incubation in 1 mM methionine). The ability of the colon to transport methionine across and concentrate methionine within its mucosa is maintained for at least 24 h after birth. Colonic transport of amino acids could be physiologically important in the pig, where the immediate post-natal transfer of immune globulins has been shown to cause a temporary inhibition of normal intestinal function.


Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures. NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/2H2O mixtures. In the lamellar Lbeta and Lgamma (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar Lalpha and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar Lalpha phase the chain is "bent" at the C9-C10 segment; but is "straight" in the hexagonal phase.


Relationship of the photosensitivity of bilayer lipid membranes and the aqueous acceptor. Studies using complex ions of amino acids. The photocurrent in photosensitive bilayer lipid membranes has been studied as a function of the aqueous acceptor. Correlations are observed between the relative photocurrent and the position of the complex ion visible absorption band and the dipole moment of the ligand. The effect of the ligands is nondirectional: they may be added to either side of the membrane with a corresponding effect on the photocurrent. The effects of the ligands are interpreted using an energy barrier model.


Protein-bound oligosaccharides of Semliki Forest virus. Semliki Forest virus was grown in BHK cells and labeled in vivo with radioactive monosaccharides. Pronase digests of the virus chromatographed on Bio-Gel P6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson, J. and Clamp, J.R. (1971) Biochem. J. 123, 739-745.) The former was labeled with [3H]fucose, [3H]galactose, [3H]mannose and [14C]glucosamine, the latter only with [3H]mannose and [14C]glucosamine. The three envelope glycoproteins E1, E2 and E3 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E1 and E3 revealed glycopeptides of A-type. E2 revealed glycopeptides of B-type. E2 yielded additionally a glycopeptide (Mr3100) which was heavily labeled from [3H]galactose, but only marginally from [14C]glucosamine, [3H]fucose and [3H]mannose. Whether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E1 and 4000 in E3; the B-type unit of E2 had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggest that E1 and E3 contain on the average one A-type unit; E2 probably contains one 3100 dalton unit plus one or two B-type units.


Isolation of the terminal complement complex from target sheep erythrocyte membranes. (1) Membranes from sheep erythrocytes lysed with antibody and human complement were solubilized in Triton X-100 and subjected to isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Membrane-bound serum proteins were located in the gels by subsequent immunoelectrophoresis against antisera to human serum proteins. Monospecific antisera against C9 and C5 were used to locate the terminal complement complex, which is not dissociated by Triton X-100. The complex focused between pH 5.8 and pH 6.5 and was separated from the bulk of other membrane-bound serum proteins, which focused at pH ranges below than 6.0. (2) In a second step, proteins electrophoretically eluted from the gel sections containing the terminal complement complex were chromatographed on Sepharose 6B equilibrated with 0.05% Triton X-100. Fused rocket immunoelectrophoresis was used to monitor separations. This step separated the terminal complement complex from the remaining contaminating proteins. The complex eluted in a broad peak corresponding to a molecular weight range of 800000-4000000. (3) The terminal complement complex thus obtained migrated with alpha-mobility and yielded a single precipitation arc in crossed immunoelectrophoresis using polyvalent antisera to human serum proteins. A distinct precipitate was obtained with monospecific anti-C9. The presence of C5 and C6, in complex with one another and with C9 was demonstrable by immuno-double-diffusion. No immunoprecipitate was obtained with antisera to sheep erythrocyte membrane proteins. (4) Dodecyl sulfate gel electrophoresis of the complex revealed seven protein bands of 190000, 160000, 115000, 93000, 85000, 68000 and 60000 daltons. Planimetric quantitation of densitometric scans gave a molar ratio of approx. 0.7:0.3:1:1:1:2:1 for these bands, respectively. All bands stained faintly with periodate-Schiff. Two-dimensional dodecyl sulfate gel electrophoresis showed that the first two bands (190000 and 160000 daltons, probably C5b and C5c) represented proteins possessing more than one peptide chain linked by disulfide bonds. The main subunit for both bands was a protein of approximately 68000 daltons. Band 5 (83000 daltons, probably C8alpha) was split into two peptide chains of approximately 68000 and 15000 daltons. The other components were not affected by dithiothreitol treatment. (5) The dodecyl sulfate gel electrophoretograms obtained were very similar to that described by Kolb and Müller-Eberhard (Kolb, W.P. and Müller-Eberhard, H.J. (1975) J. Exp. Med. 141, 724-735) for the terminal complement complex isolated from inulin-activated serum. However, certain minor but consistent deviations were observed. A preliminary correction of the electrophoretograms is presented.


Membrane and intracellular modes of sugar-dependent increments in red cell stability. Sugar-dependent increments in red cell stability under osmotic stress can be ascribed to changes either in the membrane or in the intracellular matrix. These two possible modes of action have been tested and characterized. Rheological investigation of membrane-free haemoglobin solutions has shown that D-glucose, but not D-fructose, promotes the formation of a visco-plastic gel structure. Gel strength is a function of glucose concentration, haemoglobin concentration and temperature. The ability of various sugars to promote gel formation correlates with their solution properties. The existence of gel structure reduces K+ and haemoglobin leak from red cells whose membranes were partially destroyed by gamma-radiation. Reduced osmotic swelling in the presence of glucose is also due to gel formation since the glucose effect is lost in resealed red cell ghosts. D-Fructose does not protect red cells against radiation damage; its mode of action in increasing red cell stability under osmotic stress is a membrane effect. Cell sizing using the Coulter Counter has shown that fructose, but not glucose, can increase the maximal volume at lysis. At 50 mM, D-fructose expands the red cell ghost volume by 11.2%; this represents a 7.2% increase in membrane area. Ghost expansion by fructose is fructose concentration dependent (0-100 mM) and is insensitive to temperature variation (0-37 degrees C).


Effect of a diazafluoranthen derivative on phospholipases. A study at the air-water interface. AC-3579 (2-N-methylpiperazinomethyl-1,3-diazafluoranthen 1-oxide) produces in rat hepatocytes a hypertrophy of the endoplasmic reticulum. Two possibilities that can explain this phenomenon are (1) that AC-3579 inactivates the phospholipases, and (2) that an AC-3579-lipid interaction hinders the enzymic activity. To demonstrate these hypotheses, a physicochemical model of biological membrane, the lipid-water interface, has been used. Dipalmitoyl DL-alpha-phosphatidyl-choline was spread at the air-water interface, the enzymes (phospholipase A or phospholipase C) dissolved in the aqueous phase. The enzymic reaction was first studied with and without AC-3579 dissolved in the aqueous phase; no enzymic inactivation was observed. However an AC-3579-lipid complex completely inhibited the enzymic reaction in the case of phospholipase A. An explanation is given in terms of steric hindrance to the enzyme-substrate.


The effect of sonication on the hydrocarbon chain conformation in model membrane systems: a Raman spectroscopic study. Raman spectra are presented for egg lecithin above and below the gelliquid crystal phase transition, and several regions of the Raman spectrum are shown to be sensitive to conformational changes in the hydrocarbon chains. These regions are used to investigate the effect of sonication on the structure of egg lecithin and dipalmitoyl lecithin vesicles. Sonication of both egg lecithin above Tm, and dipalmitoyl lecithin above and below Tm produces no change in the relative population of trans and gauche isomers in any of the systems studied. Sonication does however appear to effect interchain interactions, a possible consequence of imperfect packing towards the center of the bilayers in vesicle systems.


A search for a leghaemoglobin-like compound in root nodules of Trema cannabina Lour. Root nodules collected from the non-leguminous plant Trema cannabina Lour. grown under natural field conditions were examined for the presence of leghaemoglobin. No trace of any soluble haemoglobin-like compound could be detected by spectrophotometric analysis or molecular weight comparison studies, although a small amount of soluble haemoprotein with peroxidase activity was isolated. Possible alternatives to the possession of a leghaemoglobin in non-legume/Rhizobium N2-fixing associations are discussed.


Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds. A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.


Studies on the small subunit of fraction I protein from Pisum sativum L. and Vicia faba L. We have measured the molecular weight of the small subunit of Fraction I protein from pea and broad bean by sodium dodecyl sulphate polyacrylamide gel electrophoresis, Sephadex gel-filtration and amino acid composition data. The results suggest a molecular weight of 12 000-14 500, although measurements by gel-filtration in alkali suggest a molecular weight of approximately 22 000. N-terminal amino acid sequence data and C-terminal determinations show that the protein consists of a single type of polypeptide chain, although the anomalously high molecular weight obtained on gel-filtration in alkali does not preclude the existence of the polypeptides as dimers under certain conditions.


Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogenous peptides resembling collagen. To explore further the recent demonstration that hydroxyproline stabilizes the triple-helical structure of collagen, two peptides containing allohydroxyproline, (aHyp-Pro-Gly)10 and (Pro-aHyp-Gly)10, were synthesized by a modified Merrifield technique which yields products of defined molecular weight. Examination of the peptides by optical rotation and circular dichroism showed that neither of them formed triple-helical structures in aqueous solution. Since the peptides had less tendency than (Pro-Hyp-Gly)10 to become helical, the results demonstrated that the trans-4-hydroxyl group of hydroxyproline makes a specific contribution to stability of the triple helix formed by (Pro-Hyp-Gly)10. Since the peptides also had less tendency than (Pro-Pro-Gly10 to become helical, the results further demonstrated that the cis-4-hydroxyl group on allohydroxyproline decreases the stability of the triple helix. The observations provided direct support for previous data indicating that incorporation of proline analogues such as allohydroxyproline into pro-alpha chains during procollagen biosynthesis prevents the polypeptides from becoming triple helical.


A specific cephalosporin-binding protein of Citrobacter freundii. 1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium dodecyl sulfate-containing disc electrophoresis. 2. The molecular weight determined by disc electrophoresis was 53 000. 3. The binding protein did not show any beta-lactamase activity at substrate concentrations examined: 6 mM to 100 muM of penicillins and 12 mM to 100 muM of cephalosporins. 4. In gel filtration, [14C]benzylpenicillin was found not to bind to the binding protein. 5. In fluorescence titration, all cephalosporins tested quenched the fluorescence. Association constants of cephalosporins were in the range of 0.8-12-103 M-1, and one binding site was calculated for all cephalosporins tested.


Reaction of chicken egg white lysozyme with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. II. Sites of modification. 1. Procedure for the isolation of peptides from proteins bearing the chemically labile aromatic ether, O-tyrosyl-4-nitrobenz-2-oxa-1,3-diazole group, is described. 2. The tyrosyl residue reactive towards 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in chicken egg white lysozyme (Aboderin, A. A., Boedefeld, E. and Luisi, P. L., (1973) Biochim. Biophys. Acta 328. 20-30) is tyrosine-23. The amino group in the protein whose reaction with the reagent is dependent on the prior reaction of tyrosine-23 is the epsilon-amino group of lysine-33.


Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states. The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.


Purification by preparative electrophoresis and characterization of histone H2B from rat chloroleukaemia. Highly purified histone H2B from rat chloroleukaemia has been isolated by preparative electrophoresis at pH 2.7 in polyacrylamide slab gel, using the fraction F2b of Johns (Johns E. W. (1964) Biochem, J. 92, 55-59) as starting material. This histone was characterized by amino acid analysis and end groups determination. Comparative studies with homologous calf thymus histone show similarity of the amino acid compositions and of the amino terminal groups. the carboxyl terminal sequence presents two conservative substitutions.


Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation. The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and "early" fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 degrees C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.


Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family. The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.


A spectroscopic analysis of vitamin B12 derivatives. 1. Several vitamin B12 derivatives including descobalt B12 show unusual  inversion of their CD signs upon rapid cooling to liquid N2 temperature, although the room temperature CD signs are conserved by a slower cooling to the same temperature. As a possible explanation for this puzzling observation, a micro-environmental birefringence around the chromophore imbedded in an organic glass is proposed. 2. Absorption, fluorescence, phosphorescence, and polarization spectra of descobalt B12 can be correlated with those of porphyrin free bases, as these two molecular systems share many similarites in their electronic structure. 3. Molecular orbital calculations of polarization directions further support the analoby between the spectroscopic characteristics of corrins and porphyrins, and are generally in good agreement with the fluorescence polarization data.


Tissue distribution of endogenous cobalamins and other corrins in the rat, cat and guinea pig. 1. Methylcobalamin, adenosylcobalamin, hydroxocobalamin and cyanocobalamin have been estimated by a chromato-bioautographic techniques in 16 tissues from healthy rats and in five guinea pig tissues. 2. Plasma and erythrocyte cobalamins have been estimated in rats, cats and guinea pigs and the results compared with those in man. 3. Unidentified corrins were detected in 8 of the 16 rat tissues and in 3 of the 5 guinea pig tissues analysed, but were not present in tissues from specific pathogen-free rats nor in the standard laboratory diet. 4. Adenosylcobalamin was the major corrin in 8 of the 16 rat tissues. In the remainder hydroxocobalamin predominated or was present in equal proportions with adenosylcobalamin. Methylcobalamin was detected in the majority of rat tissues but at levels much lower than those in human tissues. Small amounts of cyanocobalamin were detected also and levels were higher than those of methylcobalamin in 8 of the 16 tissues. 5. In the rat, cat and guinea pig, levels of methylcobalamin and hydroxobalamin were higher in erythrocytes than in plasma, a pattern almost the complete reverse of that in man.


Isolation and characterization of a Golgi-rich fraction from the adrenal medulla. 1. A Golgi-rich fraction from bovine adrenal medulla was isolated by centrifugation through discontinuous sucrose density gradients. 2. The specific activity of UDPgalactose-N-acetylglucosamine galactosyl transferase was increased in this fraction. Therefore, this enzyme is a useful marker for Golgi in bovine adrenal medulla. 3. Golgi membranes were reasonably free from mitochondria, lysosomes, endoplasmic reticulum and chromaffin granules as shown by the relatively low activities of marker enzymes. 4. The negative staining techniques of electron microscopy revealed the presence of a system of tubules, vesicles and plate-like center regions which are similar to those structures previously described of the Golgi fraction isolated from the liver. 5. The specific activity of 5'-nucleotidase in the Golgi-rich fraction was 3.5 times greater than that in adrenal homogenates. However, the subcellular distribution patterns of galactosyl transferase and 5'-nucleotidase were similar. The possibility that 5'-nucleotidase might be a conspicious component of the Golgi apparatus is discussed.


Circadian rhythms in disaccharidases of rat small intestine and its relation to food intake. The activities of maltase and sucrase of the small intestine were low at night and high in the daytime in rats which had been fed from 09.00 h to 15.00 h for 2 weeks. A remarkable rise of enzyme activities was observed at 08.00 h, 1 h before the start of feeding. The rhythmic changes in disaccharidase activities continued for at least 2 days after  starvation, but completely disappeared after 5 days of starvation. It was suggested that the disaccharidase rhythms are not a direct consequence of food intake, but that anticipation of food intake acts as a trigger for initiation of the disaccharidase rhythms.


Metabolism of dihydroaminopterin and the influence of aminopterin and related derivatives on the metabolism of (-)-5-formyl-tetrahydrofolic acid in Pediococcus cerevisiae. [3H]Dihydroaminopterin, but not [3H]methotrexate or [3H]aminopterin, was rapidly taken up by resting cell of Pediococcus cerevisiae and inhibited the uptake of (-)-5-formyl-tetrahydrofolic acid to more than 90% at a concentration of 25 muM. On the other hand 5-formyl-tetrahydrofolic acid inhibited the uptake of dihydroaminopterin to a similar extent. Dihydroaminopterin was metabolized by Pediococcus cerevisiae, but not by Lactobacillus casei, to a compound tentatively identified as dihydroaminopterin diglutamate. Methotrexate or aminopterin at a concentration of 25 muM did not inhibit the uptake of 5-formyl-tetrahydrofolic acid in Pediococcus cervisiae, but inhibited the conversion of this compound to polyglutamate forms almost completely at a level of 2.5 mu M.


Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron. Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these iron-binding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.


Reactions of phenyldiazene and ring-substituted phenyldiazenes with ferrihemoglobin. Two simultaneous reactions take place between ferrihemoglobin and phenyldiazene in the absence of excess ferricyanide or of oxygen, namely the reduction of ferrihemoglobin to ferrohemoglobin and the binding of an exogenous ligand by ferrihemoglobin to form a compound with the optical spectrum of a ferrihemochrome. In the presence of excess ferricyanide, only the formation of a ferrihemochrome is observed. This compound differs from the ferrihemochrome induced by salicylate or by benzoate with respect to optical spectrum, concentration of inducer, and stability. One phenyl group is bound per heme, probably as phenyldiazene. Phenyl groups are also bound to globin, but phenyldiazene does not react anaerobically with thiols. Each ring-substituted isomer of methylphenyldiazene or bromophenyldiazene yields a different ferrihemochrome spectrum with ferrihemoglobin in the presence of ferricyanide. Only reduction of ferrihemoglobin occurs with 2- or 4-diazenylbenzoic acid in the absence of excess ferricyanide, but partial formation of ferrihemochrome occurs with 4-diazenylbenzoic acid in excess ferricyanide. The ability of an aryldiazene to bind quantitatively to ferrihemoglobin parallels the ability of the corresponding arylhydrazine to induce in vivo hemolysis.


Multiple forms of acetylcholinesterase from rat erythrocytes. Effect of fat-free diet. Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1--0.2%, or by ultrasound for 5 min. Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the "phenotypic allosteric desensitization phenomenon".


High affinity binding of human and bovine thrombins to p-chlorobenzylamido-epsilon-aminocaproyl agarose. Human thrombin (EC 3.4.21.5) binds tightly to p-chlorobenzylamido-epsilon-aminocaproyl agarose, and is not eluted by 2 M NaCl at pH 8. Its zymogen, human prothrombin, does not bind to the same absorbent. 2 M NaCl partially elutes DFP-treated thrombin. For native human and bovine thrombins, protein and activity are quantitatively eluted by 25% dioxane, and upon rechromatography the active human enzyme exhibits the same binding properties. Equally tight binding of human thrombin occurs with derivatives of the m- and p-chlorobenzylamines. With the o-chloro derivative or benzylamine itself insolubilized to epsilon-aminocaproyl agarose, thrombin is eluted by high ionic strength. Bovine trypsin and bovine factor Xa bind less tightly than thrombin to p-chlorobenzylamido-epsilon-aminocaproyl agarose, being eluted by high ionic strength. It is proposed that the specific thrombin adsorption is related to a secondary binding site of high affinity and with hydrophobic properties. This site is not available in the zymogen. Furthermore, the less specific protease, trypsin, and the more specific protease, factor Xa, lack this binding site.


Stabilization of glucosaminephosphate synthase from rat liver by hexose 6-phosphates. Properties and interconversion of two molecular forms. Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37 degrees C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methyl-glyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography. During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore intercovertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A. These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase.


Glucosaminephosphate synthase of human liver. Although human liver contains glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19), its activity is rapidly lost during the course of extraction. The inactivation, however, is largely prevented if the extraction medium contains isopropanol at 1% concentration; using these "stabilized" extracts, the glucosaminephosphate synthase activity of human liver has been shown to be similar to the activity previously reported in rat liver. The enzyme precipitated from these extracts by (NH4)2SO4 is inhibited by UDP-N-acetylglucosamine, the concentration required to produce a half-maximal inhibition being 6 muM. These results seem to be sufficient to postulate that glucosaminephosphate synthase is important for UDP-N-acetylglucosamine synthesis in human liver. In contrast to the rat liver enzyme, the (NH4)2SO4-precipitated human liver enzyme is resistant to trypsin and undergoes no conversion reaction when incubated with glucose 6-phosphate.


Chemical modifications of the crystalline quinolinate phosphoribosyltransferase from hog liver. Amino acid analysis and chemical modification of the crystalline quinolinate phosphoribosyltransferase (EC 2.4.2.19) from hog liver were performed. The enzyme contained 29 residues of half cystine per mol. The enzyme activity was strongly inhibited by sulfhydryl reagents. The number of reactive (exposed) sulfhydryl group was determined to be 10.2 and total sulfhydryl group was to be 25.2 per mol by using 5,5'-dithiobis(2-nitrobenzoic acid). The enzyme activity was also inhibited by lysine residue-, histidine residue-, and arginine residue-modifying reagents. These results and the effect of preincubation with the substrates on chemical modifications suggest that the lysine residue, histidine residue and sulfhydryl group may be closely related to the binding site of quinolinic acid.