Nuclear magnetic resonance studies of the solution conformation of nucleoside diphosphohexoses and their components. The solution conformations of UDPG, UDPGN, UDPGal, UDPM, UDPGluc, UDPGalc, ADPG, ADPM, GDPG, GDPM, and CDPG and their components Glu-1-P, Gal-1-P, Man-1-P, Gluc-1-P, Galc-1-P, ADP, GDP, UDP, and CDP are studied by high resolution fast Fourier transform nuclear magnetic resonance spectroscopy with iterative computer line shape simulation. The following results were observed. (1) The six-membered ring is in 4C1 chair form with the C(5')-C(6') bond in gg equilibrium tg equilibrium for the derivatives of glucose and mannose and gt equilibrium tg for those of galactose. (2) No conformational preference can be detected for C(1')-O(1') bond in hexose-1'-P moiety. (3) Chemical shift dependencies for the pyranoid ring protons and their structural and conformational relations are: (a) axial proton is at higher field than equatorial: (b) the shielding effect of a gauche vicinal hydroxyl group is stronger than a trans vicinal; (c) the vicinity of a hydroxyl group located more than three bonds away tends to shift the proton downfield. (4) The conformation of the nucleoside 5'-diphosphate part is [anti, 2E equilibrium 3E, g'g' equilibrium g't', g'g' equilibrium g''/t''], with slight variation of each conformation occuring for individual compounds. (5) No significant interactions are detected between the hexose and nucleoside parts in the nucleoside diphosphohexoses, and the hexose and nucleoside components display the same conformational preference as they become integrated to form nucleoside diphosphohexoses.


Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction. Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.


Biological activity of 1,25-dihydroxyvitamin D2 in the chick. 1,25-Dihydroxyvitamin D2 has been prepared from 25-hydroxyvitamin D2 using rachitic chick kidney mitochondria. This metabolite was highly purified by Sephadex LH-20 chromatography and by preparative high-pressure liquid chromatography. Its purity was assessed by analytical high-pressure liquid chromatography which revealed no other 254-nm absorbing material and by mass spectrometry. The concentration of dilute solutions of 1,25-dihydroxyvitamin D2 was determined by high-pressure liquid chromatography and deflection of the 254-nm column monitor. The 1,25-dihydroxyvitamin D2 was then shown to be 1/5 to 1/10 as active as 1,25-dihydroxyvitamin D3 in the chick while it had previously been shown to be equal in activity in the rat. Thus, discrimination against the vitamin D2 side chain by the chick persists in the metabolically active 1,25-dihydroxyvitamin D compounds.


Structural identification and synthesis of luciferin from the bioluminescent earthworm, Diplocardia longa. For the first time, luciferin from a bioluminescent earthworm has been purified, identified, and synthesized. This luciferin from the North American species, Diplocardia longa, is a simple aldehyde compound, N-isovaleryl-3-aminopropanal, with an amide functional group. It is a clear, odorless oil at room temperature. It is nonvolatile and has no near-uv-visible absorption or fluorescence. Derivatives of this compound were made to facilitate its identification: the luciferin 2,4-dinitrophenylhydrazone (mp 174 degrees C), a yellow crystalline solid; and the luciferin alcohol, a clear oil. Synthesis of Diplocardia luciferin yielded an oil of identical spectroscopic (proton nuclear magnetic resonance (NMR), 13C NMR, mass, and ir), chemical (dinitrophenylhydrazone and alcohol derivatives, bioluminescence activity), and physical (thin-layer chromatography, volatility) properties to those of the purified native Diplocardia luciferin.


Nucleotide sequence of phenylalanine transfer ribonucleic acid from pea (Pisum sativum, Alaska). Phenylalanine transfer ribonucleic acid from peas (Pisum sativum, Alaska) was completely digested with beef pancreatic ribonuclease (RNase I) and with ribonuclease T1. The resulting oligonucleotides were compared with those from the corresponding hydrolyses of phenylalanine transfer ribonucleic acid from wheat germ. The structures of both ribonucleic acids appeared to be identical. This report is the first to show that identical structures for the same specific acceptor transfer ribonucleic acid are present in two different plant species.


Effect of thyroid hormones on the level of the hepatic mRNA for alpha2u globulin. A procedure is presented for the purification of alpha2u globulin, a protein synthesized in the liver, secreted into the serum, and excreted in the urine of male rats. The steady-state levels of alpha2u globulin in the serum and liver cytosol fraction of adult male rats have been determined using a radial immunodiffusion assay. A cell-free protein synthesizing system, easily prepared from commercial wheat germ, has been used to identify and quantitate the mRNA coding for alpha2u globulin. Poly(A)-containing RNA isolated from male rat liver directs the synthesis in the wheat germ translational system of a protein which is precipitated by rabbit anti-alpha2u globulin and migrates with authentic alpha2u globulin on sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-containing RNA from the livers of female rats or from the kidneys of male rats, tissues which synthesize no alpha2u globulin, do not direct the synthesis of alpha2u globulin in the wheat germ system. Thyroidectomized male rats had no detectable alpha2u globulin in their sera or liver cytosols, and the livers from these thyroidectomized males were found to contain no translatable mRNA for alpha2u globulin, as measured in the wheat germ system. Administration of L-thyroxine or triiodo-L-thyronine to hyroidectomized males resulted in the synthesis of alpha2u globulin, as measured by increased levels of this protein in sera and liver cytosols. This increase in alpha2u globulin synthesis following thyroid hormone treatment was accompanied by a parallel increase in the functional level of the hepatic mRNA coding for this protein. Treatment of thyroidectomized males with a variety of androgens failed to stimulate alpha2u globulin synthesis, and no alpha2u globulin mRNA could be detected in the livers from these androgen-treated thyroidectomized males. These findings indicate that thyroid hormones influence alpha2u globulin synthesis in male rat liver by acting pretranslationally, possibly by modulating gene transcription, and rule out the possibility of an indirect androgen-mediated effect of thyroid hormones in modulating alpha2u globulin biosynthesis.


Biosynthesis of membrane bound Ig and secretion of Ig by chicken lymphoid cells. The metabolic turnover of membrane proteins of chicken lymphoid cells is studied, using a double isotope labeling technique (i.e., [14C]amino acid pulse and [3H]leucine chase). Compared with other membrane proteins, the metabolic turnover of membrane bound immunoglobulins (M-Ig) is very slow. There was no difference in the turnover between M-Ig and specific antigen binding receptor immunoglobulins. Immunoglobulins appear to be a stable constituent of the lumphocyte membrane. Cellular kinetic experiments show that the rate of biosynthesis of secreted immunoglobulins (S-Ig) is nearly ten times as much as that of M-Ig, suggesting that metabolic pathway leading to M-Ig are distinct from those leading to S-Ig. The difference in 3H/14C ratios between S-Ig and M-Ig reflects the rate of biosynthesis of these immunoglobulins by two types of bursa derived lymphoid cells.


Inhibition of chicken pyruvate kinases by amino acids. Alanine, serine, and phenylalanine behave as inhibitors competitive with phosphoenolpyruvate for the activated forms of the chicken pyruvate kinases. On the other hand, phenylalanine and alanine behave as K-type inhibitors and serine behaves as a heterotropic activator of pyruvate kinase variants which undergo homotropic activation. Tryptophan lowers the Vm and tends to yield complex plots with all variants studied. Kinetic patterns obtained in the presence of phenylalanine also show some characteristics not generally associated with a competitive mechanism. These observations are related to data previously obtained using the rat isozymes and are used to formulate a mechanism which explains the effects of the amino acids. This mechanism hypothesizes that all the effector amino acids bind to the phosphoenolpyruvate site; however, amino acids with nonpolar side chains also interact with a nonpolar region of the T conformer and thereby stabilize it. It is further proposed that there are two such nonpolar regions on the various pyruvate kinases--the one which reacts with the nonbulky side chains, and another which reacts only with relatively bulky side chains. The stabilizing effect of this second nonpolar interaction imparts inhibitory characteristics which are not competitive in nature. Serine and perhaps other polar compounds may also bind at the phosphoenolpyruvate site, but because of their polarity exert a repulsive force at the same nonpolar site with which the nonbulky nonpolar amino acids interact. This repulsion stabilizes the R conformation. Presumably the homotropic activating effects of phosphoenolpyruvate operate via this same mechanism. The data are also used to support a specific sequential-concerted mechanism for the homotropic activating effect of phosphoenolpyruvate. According to this mechanism, phosphoenolpyruvate adds sequentially to the first two subunits. This interaction causes the respective subunits to convert to the R conformation but, once two subunits are in R conformation, the remaining two subunits convert in concert.


Purification and characterization of human plasma lecithin:cholesterol acyltransferase. A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.


Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with diazonium-1H-tetrazole. Diazonium-1H-tetrazole was tested as a potential active-site-directed reagent for amino acid residues involved in catalysis by alcohol dehydrogenase. In a novel reaction with a protein, diazonium-1H-tetrazole inactivated the enzyme selectively, and almost stoichiometrically, but reacting with the sulfur of a cysteine residue, Cys-174. As a model compound, the tetrazole adduct of free cysteine was prepared. Elementary and spectral analyses of the adduct were consistent with the structure 5-tetrazoleazo-S-cysteine. The adduct absorbs light with a maximun at 316 nm, and is destroyed by irradiation at this wavelength. The inactivated enzyme still bound NADH as determined by difference spectroscopy, but did not enhance the fluorescence of the bound NADH as did native enzyme. X-ray crystallographic studies of free enzyme have shown that Cys-174 coordinates the zinc at the active site (Eklund, H., Nordström, B., Zeppezauer, E., Söderlund, G., Ohlsson, I., Boiwe, T., and Brändén, C-I. (1974), FEBS Lett. 44, 200-204). The modified enzyme is probably inactive because the large, negatively charged tetrazole ring interferes sterically or electrostatically with the binding of substrates or with hydride transfer.


Ultraviolet photoinactivation of galactosyltransferase. Protection by substrates. Galactosyltransferase was irreversibly inactivated upon exposure to ultraviolet light and the rate of inactivation followed apparent first-order kinetics. Significant protection against inactivation was observed in the presence of various combinations of substrates. UDPgalactose and Mn2+ together gave the most protection. Amino acid analyses revealed the loss of 1 mol of tryptophan per mol of galactosyltransferase upon ultraviolet photoinactivation. Further evidence for an essential trypotphan was provided by difference spectra and by inactivation with 2-hydroxy-5-nitrobenzyl bromide and protection against this reagent by Mn2+ and UDPgalactose. The protection by UDPgalactose and Mn2+ was greater than that provided by UDPgalactose alone. Since Mn2+ provided no protection by itself, this suggested that the formation of the galactosyltransferase-Mn2+-UDPgalactose complex caused a conformational change which was responsible for the observed protection of the essential tryptophanyl residue.


1H nuclear magnetic resonance double resonance study of oxytocin in aqueous solution. Peptide NH resonances in the 250 MHZ 1H nuclear magnetic resonance (NMR) spectrum of oxytocin in H2O were assigned to specific amino acid residues by the "underwater decoupling" technique (i.e., decoupling from corresponding CalphaH resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster an V. J. Hruby ((1973), Proc. Natl. Acad. Sci. U.S.A. 70, 3806) and A. F. Bradbury et al. ((1974), FEBS Lett. 42, 179). Three methods of assigning NH resonances of peptides--solvent titration, underwater decoupling, and isotopic labeling--are compared. As the solvet composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE's) of 14 +/- 5 were observed for the Tyr ortho CH and meta CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipoledipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE's therefore indicate intimate contact between water and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between water and NH group varies with Ph in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shiedled from the solvent.


Structure of the concanavalin A-methyl alpha-D-mannopyranoside complex at 6-A resolution. The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A-methyl alpha-D-mannopyranoside complex and is 35 A from the iodophenol binding site (K. D. Hardman and C. F. Ainsworth (1973), Biochemistry 12,4442), which has been postulated to be adjacent to the carbohydrate-specific binding site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 2580). The crystals are orthorhombic in space group C222(1) and crystal denisty measurements indicate a protein mass of four monomers (molecular weight of 104 000) per asymmetric unit. However, the electron density map contains eight monomers/asymmetric unit, revealing lattice disorder. The electron density map with a nominal resolution of 6 A has been solved using three heavy-atom derivatives and the position and orientation of each monomer established. Atomic coordinates of the native protein which has previously been determined (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103) were transposed into this new space group and the gross conformations of the monomers, dimers, and tetramers were found to be very similar to the previous structure. However, some minor differences were apparent even at this resolution. After crystal growth, the methyl alpha-D-mannopyranoside was replaced by o-iodophenyl beta-D-glucopyranoside or methyl 2-iodoacetimido-2-deoxy-alpha-D-glucopyranoside in separate experiments, and difference electron density maps were calculated. The highest peaks for both iodinated sugar derivatives associated with each monomer agreed within a few angstroms of each other and were found near side chains Tyr-12 and -100 and Asp-16 and -208. This region is 10-14 A from the manganese, in good agreement with nuclear magnetic resonance (NMR) studies in solution (C. F. Brewer et al. (1973), Biochemistry 12, 4448) and with the site predicted from crosslinked 1222 crystal studies (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103).


The primary structure of myohemerythrin. The complete amino acid sequence of muscle hemerythrin (myohemerythrin) from the sipunculid Themiste (syn. Dendrostomum) pyroides has been determined by analysis of tryptic, chymotryptic, and cyanogen bromide peptides. The primary structure of myohemerythrin differs substantially from that of coelomic hemerythrins of Phascolopsis (syn. Golfingia) gouldii and Themiste pyroides, the amino acid sequence of the muscle protein being only 46 and 45% homologous with the respective coelomic hemerythrins. The most extensive regions of homology between muscle and coelomic proteins occur near the terminii. These and other shorter regions of homology are interpreted in terms of the essential iron ligand residues of the active center.


Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane. Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.


Determination of the complete amino acid sequence of bovine cardiac troponin C. The amino acid sequence of bovine cardiac troponin C has been completely determined. The protein was cleaved by cyanogen bromide and the resulting peptides were isolated. All of the 161 residues of the protein could be accounted for in 12 cyanogen bromide peptides. Overlapping peptides were generated by tryptic digestion of citraconylated troponin C and isolation of the resulting five peptides. The primary structure of cardiac troponin C was elucidated by sequential manual Edman degradation of these peptides. It consists of four homologous regions, one of which probably has lost the ability to bind calcium ions. By comparing the amino acid sequence of cardiac troponin C with the sequence of skeletal troponin C, it was found that the mutation rate of the region that does not bind calcium is almost twice as high as the mutation rate of the three homologous regions that do bind calcium.


Human alpha-crystallin: characterization of the protein isolated from the periphery of cataractous lenses. alpha-Crystallin has been isolated from the peripheral region of old cataractous lenses. It was found to be closely related to bovine alpha-crystallin and to human newly synthesized alpha-crystallin in terms of its amino acid composition, the  size of its polypeptide chains and the lack of free NH2-terminal groups. However, in contrast to the simple urea gel electrophoretic polypeptide patterns obtained with the reference proteins, 11 polypeptides were detected in the preparation. Ten of the polypeptides were isolated and shown to be either A or B chains on the basis of their amino acid compositions and comparison of the peptide maps of their tryptic hydrolysates. The four B chains as well as the six A chains were closely related, with most of the tryptic peptides being common to all members of their respective group. A nomenclature based upon the urea gel electrophoretic mobilites of the polypeptides has been proposed to define each chain. It was found that this alpha-crystallin preparation is composed of at least two populations of macromolecules, one of which contains macromolecules greater than 5 X 10(6) daltons on the basis of gel filtration with Bio-Gel A-5m. The compositions of the two fractions were found to be essentially identical.


31P nuclear magnetic resonance chemical shielding tensors of phosphorylethanolamine, lecithin, and related compounds: Applications to head-group motion in model membranes. 31P nuclear magnetic resonance (NMR) powder spectra have been used to obtain the principal values of the chemical shielding tensors of dipalmitoyellecithin (DPL), dipalmitoylphosphatidylethanolamine, and several related organophosphate mono- and diesters. In addition, the principal values and orientation of the phosphorylethanolamine shielding tensor were determined from 31P NMR spectra of a single crystal. In all compounds studied the shielding tensors were clearly monaxial. The monoester spectra are typified by the spectrum of phosphorylethanolamine with principal values of -67, -13, and 69 ppm relative to H3PO4. The diesters have a larger total anisotrophy, as indicated by the DPL values of -81, -25, and 108 ppm. These data as well as the orientation of the phosphorylethanolamine shielding tensor are correlated with the electron density distribution as determined by the bonding pattern of the phosphate. The spectrum of a DPL-water (1:1) mixture at 52 degrees C has a shift anisotrophy of 30 ppm and displays a shape characteristic of an axial tensor. This change from the rigid lattice DPL pattern is explained in terms of motional narrowing, and the shielding tensor data are used to interpret the motion of the phospholipid head group. Simple rotation about the P-O(glycerol) bond is excluded, and a more complex motion involving rotation about both the P-O (glycerol) and glycerol C(2)-C(3) bonds is postulated.


Lipoteichoic acid from Bacillus licheniformis 6346 MH-1. Comparative studies on the lipid portion of the lipoteichoic acid and the membrane glycolipid. A lipoteichoic acid and a membrane glycolipid were isolated from Bacillus licheniformis 6346 MH-1. The fatty acid composition of the two preparations were similar. Most of the fatty acids were of the branched chain type. The glycolipid was shown to be a diacyl derivative of O-beta-D-glucopyranosyl-(1 leads to 6)-O-beta-D-glucopyranosyl-(1 leads to 3)-glycerol. The lipoteichoic acid contained lipid, polyglycerol phosphate, and glucosamine. The lipid was released by treatment with hydrofluoric acid and by hydrolysis in dilute acid and was shown to have a structure identical with that of the membrane glycolipid.


The polypeptide composition of ubiquinone-cytochrome c reductase (complex III) from beef heart mitochondria. The subunit structure of ubiquinone-cytochrome c reductase (complex III) has been examined and eight different polypeptides have been identified. Apparent molecular weights for each have been obtained by one or more methods including polyacrylamide gel electrophoresis in sodium doecyl sulfate and in sodium dodecyl sulfate-8 M urea and by gel filtration in sodium dodecyl sulfate and in 6 M guanidine hydrochloride. Values obtained are as follows: I, 47 500; II, 45 500; III, 29 500; IV, 27 800; V, 24 800; VI, 13 900; VII, 10 700; VIII, 4 800-9 00. Individual polypeptides have been purified and the amino acid composition of several of these have been determined. At least one polypeptide, the apoprotein of cytochrome b, is hydrophobic in character and this is a mitochondrially synthesized component (B. Lorenz, W. Kleinow, and H. Weiss (1974), Hoppe-Seyler's Z. Physiol. Chem. 355, 300). Other polypeptides including the hemoprotein of cytochrome c1 are more hydrophilic in amino acid composition.


The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I. Chemical composition and structural studies. The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety.


The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol. The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.


Immunochemical studies of human fibrinopeptide A using synthetic peptide homologues. Previous studies have indicated that rabbit antisera R2 and R33 to human fibrinopeptide A differ markedly in terms of cross-reactivity with fibrinogen and fibrinopeptide A-containing fragments of the fibrinogen molecule. Antiserum specificity was characterized by comparison of inhibition of binding to radiolabeled tyrosyl fibrinopeptide A produced by synthetic fragments and enzymatic digests of the fibrinopeptide A molecule vs. the complete fibrinopeptide sequence (Aalpha 1-16). Synthetic COOH-terminal homologues through the dodecapeptide (Aalpha 5-16) exhibited less than 16% immunoreactivity with R33 antiserum, which cross-reacts extensively with fibrinogen and fibrinopeptide A-containing fibrinogen fragments. In contrast, the synthetic COOH-terminal decapeptide (Aalpha 7-16) gave 100% immunoreactivity with R2 antiserum, which cross-reacts minimally with fibrinogen and fibrinopeptide A-containing fibrinogen fragments. Synthetic homologues smaller than Aalpha 7-16, such as Aalpha9-16 and Aalpha 7-11, reacted only minimally with R2 antiserum. Carboxypeptidase B digests of fibrinopeptide A retained less than 25% of their initial immunoreactivity with R2 antiserum. It is concluded that the antigenic determinants of R2 immunoreactivity reside entirely within the COOH-terminal ten-residue sequence of fibrinopeptide A, and that Phe-8, Asp-7, and Arg-16 contribute significantly to R2 immunoreactivity. The R2 antigenic determinants appear to be significantly less accessible to reaction with antibody than the R33 determinants when the fibrinopeptide is attached to its parent alpha chain (Canfield et al., 1976). A possible mechanism for the sequestration is discussed.


Nuclear magnetic resonance study of hydrogen-bonded ring protons in oligonucleotide helices involving classical and nonclassical base pairs. A study of the exchangeable ring nitrogen protons in aqueous solutions of oligonucleotide complexes involving Watson-Crick base pairs as well as Hoogsteen pairs and other nonclassical hydrogen bonding schemes shows that resolvable resonances in the low-field (-10 to -16 ppm from sodium 4,4-dimethyl-4-silapentanesulfonate) region can be detected in a variety of structures other than double stranded helices. Ring nitrogen proton resonances arising from the following hydrogen-bonding situations are reported: (1) AT and GC Watson-Crick base pairs in a self-complementary octanucleotide, dApApApGpCpTpTpT; (2) U-A-U base triples in complexes between oligo-U15 and AMP; (3) C-G-C+ base triples in complexes between oligo-C17 and GMP at acid pH; (4) s4U-A-s4U base triples in complexes between oligo-s4U15 and AMP, all of which involve both Watson-Crick and Hoogsteen base pairing to form triplexes; (5) C-C+ base pairing between protonated and unprotonated C residues in oligo-C17 at acid pH; and (6) I4 base quadruples in the four strand association among oligo-I at high salt. The behavior of the dA3G-CT3 helix is consistent with both fraying of the terminal base pairs and presence of intermediate states as the helix opens. In the monomer-oligomer complexes, under the conditions used here, the exchange appears to be governed by the dissociation rate of monomer from the complex. These findings suggest that those tertiary structure hydrogen bonds in tRNA involving ring nitrogen protons should have representative resonances in the low-field (11-16 ppm) proton NMR region in H2O.


Hydrogen-bonded complexes of the ribodinucleoside monophosphates in aqueous solution. Proton magnetic resonance studies. A proton magnetic resonance study of the chemical shifts of a series of ribodinucleoside monophosphates in neutral H2O solution has been recorded in the 1-100 mM concentration range. The self-complementary dinucleoside monophosphates CpG and GpC and the complementary mixture GpU + ApC form intermolecular hydrogen-bonded complexes at low temperatures. The amino proton chemical shifts in the CpG and GpC spectra are consistent with the formation of a miniature double helical dimer in neutral aqueous solution at low temperatures (approximately 2 degrees C). The complementary mixture of dinucleosides GpU + ApC formed much less stable complexes than either GpC or CpG, while UpA did not show any indication of the formation of intermolecular hydrogen-bonded complexes. This result is consistent with the well-known observation that the stability of a double helix is proportional to the percent of G-C base pairs present.


Study of triple-singlet energy transfer in an enzyme-dye complex using optical detection of magnetic resonance. We have made optical detection of magnetic resonance (ODMR) measurements on the enzyme alpha-chymotrypsin, as well as on its complex with the dye, proflavin. Evidence that triplet-singlet energy transfer occurs in the complex is provided by the observation of characteristic tryptophan ODMR signals while monitoring the delayed fluorescence of the dye. The luminescence decay kinetics of the complex indicates that nontrivial triplet-singlet transfer originates from several (at least three) tryptophan residues of the enzyme. ODMR sensitivity can be enhanced by coupling the sublevels of a weakly radiative triplet state to a fluorescent dye which satisfies Förster's (Förster, T. (1948), Ann. Phys. (Leipzig) 2, 55; (1965), in Modern Quantum Chemistry, Istanbul Lectures, Part III, Sinanoglu, O., Ed., New York, N.Y., Academic Press, p 93) conditions for energy transfer.


Mutant forms of cytochrome P-450 controlling both 18- and 11beta-steroid hydroxylation in the rat. A reciprocal relationship between steroid 18- and 11beta-hydroxylase activities in the salt susceptible (S) and the salt resistant (R) strains of rats was previously shown to be controlled by a single genetic locus with two alleles and inheritance by co-dominance (Rapp, J. P., and Dahl, L. K. (1972), Endocrinology 90, 1435). The strain specific steroidogenic patterns, characterized by the relative magnitudes of 18- and 11beta-hydroxylase activities, were found to be determined by adrenal mitochondrial cytochrome P-450 particles. Carbon monoxide inhibition of 18- and 11beta-hydroxylation of deoxycorticosterone in these strains showed that the CO/O2 ratio causing 50% inhibition (i.e., Warburg's partition constant, K) was identical for 18- and 11beta-hydroxylation within a strain, but different for both 18- and 11 beta hydroxylation between strains. (K values were: S rats, 18-hydroxylation = 11.4 +/- 1.4; S rats, 11beta-hydroxylation = 11.0 +/- 1.2; R rats, 18-hydroxylation = 56.4 +/- 13.7; R rats, 11beta-hydroxylation = 46.7 +/- 11.7). This between-strain difference was unique for 18- and 11beta-hydroxylation; i.e., it was not seen with cholesterol side-chain cleavage or 21-hydroxylation. Moreover, the strain-specific K values for 18- and 11beta-hydroxylase and the strain-specific steroidogenic patterns due to the relative magnitudes of 18- and 11beta-hydroxylase activities segregated together in an F2 population. These data strongly suggest the same cytochrome P-450 is involved in both 18- and 11beta-hydroxylation and that this cytochrome is mutated between S and R rats. K values for the reaction corticosterone leads to 18-hydroxycorticosterone were different between S and R strains, indicating that the mutant cytochrome was also involved in this hydroxylation, but K values for the conversion corticosterone leads to aldosterone were not different between strains. This was interpreted to mean that each step in the sequence corticosterone leads to 18-hydroxycorticosterone leads to aldosterone was mediated by a different cytochrome, the K value for the second step being the lower and dominating the overall reaction. It was speculated that the second step could be a second hydroxylation at position 18 to yield 18,18-dihydroxycorticosterone which could be unstable and decompose into aldosterone and water.


Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phophoslipid artificial membranes. The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles is a function of the physical state of the lipid. Below the phase transition, the polarization approaches the theoretical maximum for total immobilization while above the phase transition the fluorescence becomes nearly completely depolarized. The discontinuity in the temperature dependence of polarization occurs within a temperature range under 5 degrees C in the case of pure phospholipids, but for mixed phospholipids occurs over a temperature range greater than 20 degrees C. From these data, phase diagrams describing the gel-sol equilibrium can be constructed; the phase diagrams correspond well with those described in the literature which were constructed using spin-label probes or from x-ray diffraction patterns. The marked change in polarization at the phase transition may be related to the packing of the probe molecule into the lipid bilayer: fluorescence measurements on oriented bilayers indicate that below the phase transition the long axis of the probe is oriented perpendicular to the plane of the membrane while above the transition the probe is oriented randomly relative to the plane of the membrane.


Transfer of xylose to steroids by rabbit liver microsomes. Rabbit liver microsomal preparations can transfer xylose from UDP-xylose to estron, 17alpha-estradiol, and 17beta-estradiol, and, in poorer yield, to diethylstilbestrol and p-nitrophenol. No transfer of xylose to estriol, testosterone, epitestosterone or 17alpha-estradiol 3-glucuronide could be demonstrated. The xyloside of [6,7-3H]estrone which was formed by liver microsomes crystallized to constant specific activity with estrone beta-D-xylopyranoside, the chemical preparation of which is described.


Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts. Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.


Chemical-enzymatic replacement of Ile64 in the reactive site of soybeen trypsin inhibitor (Kunitz). All the reactive amino groups in soybean trypsin inhibitor (Kunitz) were protected by guanidination of 9 out of 10 lysyl residues with O-methylisourea and by carbamoylation of the NH2 terminal Asp with potassium cyanate. This derivative was converted to modified inhibitor (Arg63-Ile64 reactive site peptide bond hydrolyzed) by incubation with trypsin at pH 3. The NH2 terminal of Ile64 was allowed to react with phenyl isothiocyanate to produce inactive phenylthiocarbamoyl-modified inhibitor. Treatment with trifluoroacetic acid formed the anilinothiazolinone of Ile64 yielding des-Ile64-modified inhibitor. After renaturation and purification, this material coelectrophoresed with modified inhibitor but did not form a stable complex with trypsin. Incubation with tert-butyloxycarbonyl-(amino acid)-N-hydroxysuccinimide esters yielded [tert-butyloxycarbonyl-(amino acid64)]-modified inhibitor. The tert-butyloxycarbonyl protective group was removed in trifluoroacetic acid. After renaturation, active [amino acid64]-modified inhibitors were obtained for Ile64, Ala64, Leu64, and Gly64 replacements. The resynthesis of the reactive-site peptide bound by kinetic control dissociation of the trypsin-inhibitor complex yielded fully active [Ala64]-virgin inhibitor. Thus, soybean trypsin inhibitor (Kunitz) has been shown to tolerate the replacement of the P1' residue with retention of activity. The importance of P1' residues in the function of protein proteinase inhibitors is discussed.


Chemical-enzymatic insertion of an amino acid residue in the reactive site of soybean trypsin inhibitor (Kunitz). Modified (Arg63-Ile64 reactive-site peptide bond hydrolyzed) soybean trypsin inhibitor (Kunitz) with all reactive amino groups, except that of Ile64, protected was described in the preceding paper (Kowalski, D., and Laskowski, M., Jr. (1976), Biochemistry, preceding paper in this issue). Treatment of this inhibitor with tert-butyloxycarbonyl-Ala- and tert-butyloxycarbonyl-Ile-N-hydroxy-succinimide esters yields inactive endo-tert-butyloxycarbonyl-Ala63A-and endo-tert-butyloxycarbonyl-Ile63A-modified inhibitors. The tert-butyloxycarbonyl groups were removed by treatment of the proteins with trifluoroacetic acid. After renaturation and purification, the resultant endo-Ala63A- and endo-Ile63A-modified inhibitors co-electrophorese with modified inhibitor both on disc gels (pH 9.4) and sodium dodecyl sulfate gels (after reduction of disulfide bonds) and show end groups corresponding to the 63A residue. These derivatives fail to form stable complexes with trypsin, extending the previous observation (Kowalski, D., and Laskowski, M., Jr. (1972), Biochemistry 11, 3451) that acylation of the P1' residue in modified inhibitors leads to inactivation. However, the incubation of endo-Ala63A- and endo-Ile63A-modified inhibitors with trypsin at pH 6.5 leads to the synthesis of the Arg63-Ala63A and Arg63-Ile63A peptide bonds in 4% yield. This is very close to the yield anticipated from a semiquantitative theory for the value of the equilibrium constant for reactive-site peptide bond. An alternative chemical method of insertion is also described. Controlled treatment of modified inhibitor with the N-carboxyanhydride of Glu produced inactive endo-Glu63A-modified inhibitor. Incubation of this inactive derivative with trypsin at pH 6.5 leads to 16% synthesis of the Arg63-Glu63A peptide bond. The higher yield of single chain protein in this case is attributed to the influence of the negative charge of the Glu63A side chain. Thus, the insertion of an amino acid residue between the P1 and P1' residues in soybean trypsin inhibitor (Kunitz) converts a trypsin inhibitor into a trypsin substrate.


The effect of Raney nickel on the covalent thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate complex. Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex.


The effect of opposing effectors on activation level of succinate dehydrogenase: equilibrium and kinetic studies. The activation of mitochondrial succinate dehydrogenase by various activators is a result of dissociation of oxaloacetate tightly bound to the nonactive enzyme. But, quantitative correlation between the effector concentrations and the active fraction of the enzyme was not at hand. In this study we measured the level of active succinate dehydrogenase equilibrated with a wide range of opposing effectors: oxaloacetate (1-500 muM) and activator (0.02-1.5 M NaBr). The results are compatible with a model assuming two stable forms of the enzyme: a nonactive enzyme-oxaloacetate complex and an active enzyme free of oxaloacetate. The active form is stabilized by binding two Br- and one H+. The rate of activation (ka) and exchange between enzyme bound and free oxaloacetate k(ex) were measured.  Both ka and kex are hyperbolically dependent on Br- concentration but differ in magnitude and pH dependence. kex at infinite Br- concentration is pH dependent but ka is not. The two reactions, activation and exchange, also differ in their activation energy bein 32 and 21.5 kcal/mol, respectively. It is concluded that, in the course of activation, Br- interacts at two distinct steps. First to produce a ternary, nonactive [enzyme-oxaloacetate-Br-] complex. From this complex, oxaloacetate dissociates and the oxaloacetate-free enzyme assumes its active form. Finally, the active enzyme is stabilized by binding another Br-. The rate-limiting step in deactivation is binding of oxaloacetate to active enzyme. The complex formed undergoes a very rapid transformation to the stable nonactive form. This pathway, under certain conditions, can reverse its direction and contribute to the overall rate of activation. It is suggested that the equilibrium between the two stable forms of the enzyme can be reached by two parallel pathways, each contributing independently to the observed rate of activation, while the final equilibrium is determined by the free energy between the products and the reactants.


Birefringence of glycerinated crab muscle fiber under various conditions. By using glycerinated single fibers of crab muscle (Sesarma haematocheir) which has long sarcomeres, the birefringence of the I band, H band and the overlapping region between thin and thick filaments was measured separately, under various environmental conditions. At the resting length, the birefringence of the fiber was decreased by the addition of Ca2+ in the absence of ATP, by about 0.35%. This birefringence decrease was found to take place in the overlapping region. The decrease corresponded to about 2% of the birefringence of thin filaments in this region. The birefringence of the fiber was increased by the addition of ATP in the absence of Ca2+, by about 6%. This birefringence increase also took place mostly in the overlapping region. The increase of birefringence by pyrophosphate was about half of that by ATP. The birefringence of the fiber was decreased by the increase of the ionic strength from 0.12 to 0.20. The origin of the observed changes of birefringence is discussed.


Studies on phytohemagglutinins. XXVII. A study of the pea lectin binding site. Under defined mild conditions the reaction of the pea lectin with 2-nitrophenylsulfenyl chloride results in sulfenylation of only 2 of the 10 tryptophan residues of the lectin molecule with simultaneous loss of biological activity. Both sulfenylated tryptophan residues belong to the two heavy subunits of the lectin. Enzymic hydrolysis and separation of the tryptic peptides yields only one homogeneous yellow peptide containing the modified tryptophan residue. The isolated peptide has the following sequence (NPS, nitrophenylsulfenyl): HAsp-Val-Val-Pro-Glu-(2-NPS-Trp)-Val-ArgOH. The octapeptide is either directly a part of the pea lectin binding site or it plays an important role in maintaining the tertiary structure of the binding site. According to the amino acid composition and amino acid sequence, the octapeptide isolated from the pea lectin is almost identical with that part of the peptide chain of concanavalin A near to which the location of the sugar binding site is supposed to be.


Factors affecting the properties of insolubilized anitbodies. IgG separated from an antiserum to estradiol was coupled under various experimental conditions to Sepharose activated either with CNBr or by conversion into a long-armed derivative (the N-hydroxysuccinimide ester). The conjugates were characterized by measurement of the binding parameters, in order to evaluate separately the loss of sites and the loss of affinity. The cross-reactivity with estriol and estrone was measured to obtain information on the occurrence of structural alterations of the antibody site. The results show that the loss of immunoreactivity varies in extent (from 95% to less than 10%) and in nature (loss of sites or of affinity or a combination of both effects) depending on the coupling conditions. The use of a hydrocarbon extension to keep the protein distant from the matrix does not prevent the loss of active sites but is effective in safeguarding the affinity of the residual sites. The loss of sites can be substantially reduced by coupling at a pH value around neutrality and by keeping the protein/matrix mass ratio low. At a coupling pH of 6.4 and at a mass ratio of 0.1-0.2 nmol IgG/mg of Sepharose, the antibodies were insolubilized with a negligible loss of sites and affinity; on increasing the mass ratio (up to 10 nmol IgG/mg Sepharose) there is a progressive loss of sites accompanied by a substantial lowering of the affinity of the residual sites. On the basis of the above-mentioned findings, the nature of the effects occurring when antibodies are transferred from solution onto a solid matrix is discussed.


Beta-3-Oxindolylalanine: The main intermediate in tryptophan degradation occurring in acid hydrolysis of protein. Under hydrolytic conditions using 6 M HCl, tryptophan reacted separately with dithiodiglycolic acid and cystine to give beta-3-oxindolylalanine (beta-[3-(2-indolinone)]alanine) as the main product. A compound, which eluted in the amino acid analyzer at the same position as beta-3-oxindolylalanine, was found in the acid hydrolyzate of lysozyme. The identicalness of these two compounds was established by comparison of their ultraviolet absorption spectra, elution positions on ion exchange chromatograms, etc. The "acid degradation product of tryptophan", which is known to be produced upon acid hydrolysis of tryptophan-containing proteins, must also be the same compound.


The iron and subunit binding sites of hemerythrin. The role of histidine, tyrosine and tryptophan. Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pk of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin. Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association. Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron.


Further characterization studies of the alpha-amylase protein inhibitor of gel electrophoretic mobility 0.19 from the wheat kernel. A highly purified amylase protein inhibitor from the kernels of hexaplois wheat, designated 0.19 according to its gel electrophoretic mobility, has been characterized according to its circular dichroism spectra determined at different pH values and in the presence or absence of dissociating and reducing agents. The 0.19 albumin has also been characterized according to the specificity with which it inhibits 21 alpha-amylases from different origins and according to its sensitivity to a number of chemical and enzymatic treatments of its inhibitory action on human saliva and Tenebrio molitor L. larval midgut alpha-amylases. Inhibitory activity of 0.19 toward human saliva amylase significantly increased when the inhibitor was incubated with the enzyme before the addition of starch, but it was not affected by the preincubation of 0.19 with starch. Maltose reversed the inhibition of human saliva by 0.19 and showed some inhibitory activity toward the enzyme. However, maltose concentrations that only slightly affected amylase activity were very effective in restoring the amylase activity inhibited by 0.19. The inhibitory action of 0.19 on human saliva and T. molitor L. amylases were equally resistant to trypsin and thermal treatments, but 0.19 was readily inactivated by incubation with pepsin or by reduction of disulfide bonds. The inhibition of the mammalian amylase by 0.19 was adversely affected by a treatment with CNBr (1:100 ratio of methionine residues to CNBr) whereas the inhibition of the insect amylase was not. As shown by circular dichroism measurements in the far ultraviolet, 0.19 is a protein with about 50% of ordered structure. Significant and largely reversible changes have been observed in the aromatic CD spectrum of 0.19 at alkaline pH values or in the presence of sodium dodecyl sulfate. These changes, which were associated with a partial loss of inhibitory activity, indicate that ionizable tyrosine groups contribute significantly to the ellipticity bands of 0.19 in the near ultraviolet.


Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom. By means of CM-Sephadex C-50 column chromatography, Trimeresurus mucrosquamatus venom was separated into twenty fractions. The fibrinogenolytic activity was concentrated in Fractions 8, 10, 12, 13 and 14. Fractions 8 adn 13 had the highest ratio of fibrinogenolytic and caseinolytic activities. Fraction 8 possessed tosyl-L-arginine methyl esterase activity, while the others did not. The caseinolytic activities of Fractions 10, 12, 13 and 14 were inhibited by EDTA, while that of Fraction 8 was not. Fractions 8 and 13 were further purified by CM-cellulose and gel filtration and were homogeneous as judged by electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weights of the purified Fractions 8 and 13 were 26 000 and 22 400, respectively. Both were single peptide chains. The specific fibrinogenolytic activity of Fraction 8 was 17 mg fibrinogen/min/mg protein, while that of Fraction 13was 100 mg fibrinogen/min/mg protein. Fraction 13 digested specifically the alpha(A) chain of monomeric fibrinogen to yield two cleavage products. Fraction 8 digested the beta(B) chain first to yield four cleavage products. When the incubation time was prolonged, the alpha(A) chain was also partially digested by Fraction 8 to yield two cleavage products.


Affinity chromatography of amine oxidase from Aspergillus niger. Omega-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of epsilon-aminopentyl-Sepharose or delta-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 A) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to omega-aminohexyl-Sepharose. It was also demonstrated that the affinity chromatography on omega-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.


Study of the apoprotein of Folch-Pi bovine proteolipid. II. Characterization of the components isolated from sodium dodecyl sulfate solutions. Acrylamide gel electrophoresis in dodecyl sulfate solutions of Folch-Pi apoprotein shows several bands. The different components were separated by Biogel P-200 filtration and then reduced and carboxymethylated. A comparative study of the amino acid composition, N-terminal sequence and C-terminal amino acid of the different components led to the assumption that their primary sequences are similar. Evidence for a contamination of the protein by free amino acids might explain the difference in terminal groups found by us and by other groups. It has been shown that the purified components can polymerize independently of S-S bond formation or exchange. The polymerization products were found to resist dissociation by dodecyl sulfate. It has been suggested therefore that the differences in migration rates of the various components are related to their shape rather than to their molecular weight.


The myoglobin of the Cape hunting dog (Lycaon pictus). Tryptic and other peptides from the myoglobin of the Cape hunting dog (Lycaon pictus) have been aligned with the sequence of the myoglobin of the domestic dog. One amino acid difference was found which was confirmed by dansyl-Edman degradation. The five carnivore myoglobins now known have been integrated into an evolutionary cladogram, trying to trace the pathways of mutations, and a possible ancestral myoglobin for the carnivores has been constructed.


The covalent structure of dog myoglobin. The primary structure of the myoglobin of the domestic dog (German shepherd) was studied. Tryptic and thermolytic peptides were compared with the sequence of other known myoglobins; the stepwise automatic Edman's degradation of the whole globin and also the chymotryptic digestion of the median fragment obtained by CNBr cleavage completed this sequence. Comparison of the established dog myoglobin structure with those from other carnivora shows 16 differences versus badger, 20 versus harbour seal and 15 versus California sea lion.


The nature of alkylurea and urea denaturation of alpha-chymotrypsinogen. The optical rotatory dispersion of alpha-chymotrypsinogen in aqueous solution became less levorotatory upon the addition of ethyl-, propyl-, or butylurea; less negative values for the Moffitt-Yang parameter, alphao, were also obatined. This change in optical rotation of alpha-chymotrypsinogen induced by the alkylureas was similar in direction and magnitude to that observed for alcohols but was opposite to that caused by unsubstituted urea. It appears, therefore, that the alkylureas share with the alcohols an ability to rearrange alpha-chymotrypsinogen into a non-native yet regularly ordered conformation. The effectiveness of the alkylureas and alcohols as denaturants for this protein increased in the order ethyl less than propyl less than butyl derivatives. An identical rank-order was observed for the ability of the alkylureas and alcohols to diminish attractive forces between aliphatic groups, as measured by a model system based upon the extent of aggregation of glass beads coated with methyl groups. These findings indicate that the denaturing action of alkylureas for alpha-chymotrypsinogen is a function of the substituted aliphatic group and is predominantly hydrophobic in character. Non-hydrophobic interactions of unsubstituted urea with alpha-chymotrypsinogen appear to be critical for unfolding of the protein to a random-coil configuration.


N-terminal peptide sequence of goat-beta-lactoglobulin. Three cyanogen bromide peptides from native goat beta-lactoglobulin have been isolated by gel-filtration. The N-terminal fragment has been identified and its sequence was determined to be: Ile-Val-Thr-Gln-Thr-. The results are compared with the N-terminal region of cow beta-lactoglobulins A and B.


Band-shape analysis and resolution of electronic spectra of pyridoxal phosphate and other 3-hydroxypyridine-4-aldehydes. The electronic absorption spectra of individual ionic forms of pyridoxal phosphate and of a series of related aldehydes have been evaluated together with pKa values. Spectral resolution with lognormal curves has permitted the quantitative description of equilibria for hydration and tautomerization. Precise values of peak positions for both aldehyde and hydrate forms have been obtained. Measurements of temperature-induced changes in the spectra have provided additional information. Knowing the hydration ratios and stepwide acid dissociation constants, it is possible to evaluate microscopic acid dissociation constants for both the aldehyde and hydrate forms of the compounds.


Chemical and biological properties of a peptido-glucan fraction from Armillaria mellea (Basidiomycetes). A peptide-glucan fraction from Armillaria mellea (Basidiomycetes) was isolated and some aspects of its chemical structure were determined. The glucan is linked to the peptide portion which represents 30% w/w of the complex. Treatment with alkali destroys most of the threonine and leads to the separation of the peptide and carbohydrate moieties indicating the involvement of the hydroxyl group of threonine in the peptide-glucan linkage. The results of partial hydrolysis, methylation studies and Smith degradation involving periodate oxidation, borohydride reduction and acid hydrolysis indicate that the polysaccharide moiety consists of beta(1 leads to 3) and beta(1 leads to 6)-linked D-glucose residues. This peptide-rich glucan fraction showed a significant antitumor activity.


The effect of cycloheximide on galactosyltransferase of rat liver Golgi membranes. An inhibitory effect of cycloheximide on the initial rate of galactosyltransferase of rat liver Golgi membranes has been demonstrated. Cycloheximide was effective in inhibiting the activity of the enzyme when added directly to the assay medium or after pre-incubation of the membranes with the drug. The inhibition observed with different concentrations of nucleotide sugar was shown to be competitive at higher concentrations of the nucleotide sugar (0.10-1.0 mumol). The inhibition observed with different concentrations of acceptor, N-acetylglucosamine was complex and could not be analysed further with the present data. Washing the Golgi membranes previously incubated with cycloheximide with water failed to reverse the inhibition. Washing with UDPgalactose partially reversed the inhibition only. These results, together with the observation that serum galactosyltransferase was not inhibited by cycloheximide supported the view that the cycloheximide effect may be primarily on the membrane system.


Hypertrophy and hyperplasia of the neonatal rat exocrine pancreas induced by orally administered soybean trypsin inhibitor. Measurement of RNA, DNA, protein and phospholipid synthesis in the neonatal rat pancreas following the oral administration of partially purified soybean trypsin inhibitor demonstrates an enhanced synthesis of all these constituents. Evidence of true hyperplasia in addition to this cellular hypertrophy is provided by an increased mitotic activity in the exocrine pancreas following a wave of enhanced [3H] thymidine incorporation into DNA. Complete inhibition of the stimulated RNA synthesis by low doses of actinomycin D indicates the importance of transcription as a regulatory step in the response of the exocrine pancreas to trophic stimulation by this means. Collateral observations of [3H] thymidine and [14C] orotic acid incorporation into liver DNA and RNA, respectively, fail to demonstrate comparable changes confirming the probable specificity of the trypsin inhibitor induced effect on the exocrine pancreas. It is suggested that the pronounced trophic effect of orally administered soybean trypsin inhibitor in the neonatal rat pancreas may serve as a useful model for the study of regulatory mechanisms of nucleic acid and protein synthesis in the mammalian pancreas.


Interpretation of the rate of density labelling of enzymes with 2H2O. Possible implications for the mode of action of phytochrome. The interpretation of the rate of density labelling of enzymes (the differential or comparative density labelling technique) is considered in terms of two simple models of enzyme turnover. On the basis of this illustrative analysis it is demonstrated that the interpretation of differential (comparative) density labelling experiments is critically dependent on the establishment of a definitive mechanism of enzyme turnover for each particular enzyme studied. In the light of these findings the interpretation of comparative (differential) density labelling experiments appertaining to the mechanism of phytochrome-mediated increases in enzyme activity in higher plants is re-assessed.


Effects of kinetin on phosphorylation of leaf membrane proteins. Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.


Preparation and isolation of a taste bud-derived fraction from bovine circumvallate papillae. A crude extract from taste buds was prepared from circumvallate papillae of bovine tongues by a procedure consisting of freezing, coring, excision, treatment with hypotonic buffer, nitrogen pressurization and selective homogenization. Examination of taste buds by light and electron microscopy before and after this procedure indicated that some of the contents of the buds, mainly from the more apical portions, was extruded following the procedure; anatomical changes could not be observed in the epithelial tissue immediately surrounding the taste buds.


[Study of the formation of N-glycolylmuramic acid from Nocardia asteroides (author's transl)]. Nocardia asteroides was grown in Sauton medium containing sodium [carboxy-14C]acetate. The biosynthesis of the peptidoglycan was inhibited by adding penicillin or phosphonomycin to the growth medium. These antibiotics give an accumulation of radioactive nucleotidic precursors of the peptidoglycan. In the presence of penicillin, there was an accumulation of uridine diphosphate-N-glycolylmuramyl peptide (UDP-MurNGlyc peptide) and of a mixture of uridine diphosphate-N-acetyl and N-glycolylmuramic acid (UDP-MurNAc) and UDP-MurNGlyc). In the presence of phosphonomycin, the biosynthesis of muramic acid was blocked and there was an accumulation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-N-glycolyglucosamine (UDP-GlcNGlyc). Thus the formation of a N-glycolyl group can be performed upon the neucleotidic derivatives of glucosamine and muramic acid. However in the peptidoglycan synthesized in vivo in the absence of antibiotic, only muramic acid was glycolyated. So, glycolylation seems to take place essentially on UDP-MurNAc. When the binding of peptide chain to muramic acid is achieved, all the muramic acid is glycolylated, then the polymerisation of glycan and peptidoglycan units by the mean of particulate enzymes is carried out on the N-glycolylated derivative of muramic acid. A cell-free preparation from Nocardia asteroides was obtained which can hydroxylate the acetyl group of UDP-MurNAc. The activity was localised in the soluble fraction. This system acts as a hydroxylase and requires the presence of NADPH.


Direct determination of the stoichiometry of charge transfer complexes. The stoichiometry of the charge transfer complex between N-acetyl-L-tryptophan and 1-methylnicotinamide chloride has been determined to be precisely 1:1 by direct measurement of the molecular weight of the complex. The result is of interest both in terms of a general method for determining the stoichiometry of charge transfer complexes, and in terms of the probable stoichiometry of specific charge transfer complexes between 1-methylnicotinamide chloride and the exposed tryptophyl side chains of certain proteins. In the latter case, the result provides experimental proof for the assignment of extinction coefficients of specified magnitudes to the homomorphic model complexes which serve as the basis for the interpretation of results with proteins.


Characterization, distribution and biosynthesis of the major ganglioside of rat intestinal mucosa. The major sialic acid containing glycolipid has been isolated from rat intestinal mucosa. Characterization of this ganglioside by thin layer and gas chromatographic analysis indicates that it is an hematoside (GM3) with the major portion of the sialic acid in the N-glycolyl form. The distribution of this ganglioside was determined in villus and crypt cells isolated from rat intestine. The hematoside content of crypt cells was found to be significantly decreased when compared to villus cells. CMP-sialic acid:lactosylceramide sialyltransferase, responsible for the sialylation of lactosylceramide, was measured in differentiated villus and undifferentiated crypt cells and found to be greatly reduced in the crypt cell fraction. The present study demonstrates that marked differences in ganglioside content and biosynthesis occur in contiguous populations of cells in varying states of differentiation when isolated from normal rat intestine.


The enzymic formation of long chain aldehydes and alcohols by alpha-oxidation of fatty acids in extracts of cucumber fruit (Cucumis sativus). 1. An enzyme system that catalyses the alpha-oxidation of fatty acids to shorter chain products is present in acetone powders of cucumber fruits. 2. In the absence of NAD+, the predominant product from palmitic acid is pentadecanal. Addition of NAD+ gives rise to a homologous series of n-alkanals, the concentrations of which are in the same order as that reported in the volatile products formed on homogenization of cucumbers, i.e. C15 greater than C14 greater than C13 greater than C12. 3. Pentadecan-1-ol is also formed from palmitic acid in the absence of added NAD+; C15, C14 and C13 n-alkanols are produced in the presence of NAD+. 4. The substrate specificity for saturated fatty acids is in the order C12 less than C14 greater than C16 greater than C18. Unsaturated C18 acids are oxidized more readily than stearic acid. 5. The alpha-oxidation system is inhibited by dithiothreitol, cysteine, imidazole and certain metal ligands (CN-, N3-, diphenylthiocarbazone) but not by EDTA. 6. Differences between the alpha-oxidation system in cucumber and those previously reported in other plants are discussed.


Influence of environmental temperature on the fatty acid desaturation and elongation activity of fish (Pimelodus maculatus) liver microsomes. The effect of environmental temperature on the activity of liver microsomes of fish (Pimelodus maculatus) to desaturate and elongate oleic, linoleic and alpha-linolenic acids was studied. It was found that: 1. Fish kept at 14-15 degrees C had higher desaturation and elongation activity than animals kept at 29-30 degrees C. The ratio of activity was the same for the three fatty acids. 2. A decrease of the environmental temperature increased the V of linoleic acid desaturation to gamma-linolenic acid, but did not modify the approximate Km of the reaction. 3. The inactivation of the delta6-desaturase of microsomes separated from fish kept at 29-30 degrees C and 14-15 degrees C was the same when heated at 40 degrees C. However, the enzyme was deactivated faster when heated at 29-30 degrees C than at 14-15 degrees C. 4. The increase of the delta6-desaturation activity of the microsomes evoked by the decrease of the temperature of the aquarium was mostly compensated for by the correlative decrease of the specific reaction rate of the reaction. For this reason it is assumed that the adaptive change of the desaturation activity of the microsomes with the environmental temperature does not greatly modify the fatty acid composition of the fish.


[Beta-Hydroxybutyrate dehydrogenase of rat liver inner mitochondrial membrane. Its isolation, characterization, and reactivation by lecithins differing in their apolar regions. The influence of the addition of cholesterol on its level of reactivation (author's transl)]. The beta-hydroxybutyrate dehydrogenase has been isolated and purified from the inner mitochondrial membrane of the rat liver. It consists in a dimer of molecular weight 77 000 composed by two subunits of molecular weight 38 000 each. The level of its reactivation by lecithin is influenced by the length and degree of unsaturated of their aliphatic chains. The addition of cholesterol inhibits the reactivation.


The metabolism of (1-14C) stearic acid in rat testicular tissue. (1) The metabolism of stearic acid was studied in vivo following intratesticular injection of [1-14C] stearate. Soon after injection 14C activity was found mainly in the free fatty acid pool. This was followed at later time periods by transfer of label primarily to the phosphatide pool. During each time period significant amounts of label were recovered at 14CO2. (2) Analysis of 14C-labeled fatty acids from the injected testes demonstrated an initial rapid rate of oxidation and desaturation of [1-14C] stearate followed by a slower steady state rate. It was concluded that the initial rate was due to the rapid turnover of the highly labeled free fatty acid pool followed by a much slower rate as [14C] stearate was esterified to the more metabolically stable phospholipids. Elongation of the labeled stearic or its desaturated derivative was not observed. (3) The rate of desaturation in vitro of stearic acid was measured in microsomal preparations from rat testes and found to be 12.0 +/- 0.5 pmol/min/mg compared to the estimated in vivo value of 22 pmol/min/mg and the value of 390 pmol/min/mg for hepatic microsomal desaturase.


Nuclear magnetic resonance studies of the aggregation of dihexanoyllecithin and of diheptanolyllecithin in aqueous solutions. Aggregation of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (dihexanoyllecithin) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (diheptanoyllecithin) in aqueous solutions has been investigated by 1H nuclear magnetic resonance spectroscopy. The chemical shifts and line widths of the NMR signals of the lecithins are dependent on the total concentration of lecithin above the critical micelle concentration. Signals for both lecithins in the aggregated state exhibit line widths which are appreciably smaller than the dipolar line width calculated using the overall rotational correlation time of the micelle. Signals of the alpha-methylene protons of the carboxylic acid side chains of dihexanoyllecithin and diheptanoyllecithin undergo the greatest change in chemical shift on aggregation. A single averaged spectrum of the alpha-methylene protons is observed in lecithin solutions of concentrations ranging from one to four times the critical micelle concentration demonstrating that individual lecithin molecules are in rapid exchange, with respect to a frequency of 18 Hz, between the monomeric and the aggregated states. Plots of the chemical shift of the alpha-methylene protons versus concentration of lecithin approximate a micelle formation curve. At about five times the critical micelle concentration for both dihexanoyllecithin and diheptanoyllecithin the alpha-methylene pattern indicates that there are at least two magnetic environments for lecithin molecules in the aggregated state. Furthermore, individual lecithin molecules are in slow exchange between the two environments which are distinguished by a chemical shift difference of about 2 Hz.


In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats. 1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.


Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis. Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.


Sphingolipids of influenza viruses.

Sphingolipids of influenza viruses. Total lipid of four egg grown influenza viruses (A2-Asia, A2-England, A2-Taiwan and fowl plague virus) were extracted with chloroform-methanol. After mild alkali treatment of the extracts, glycosphingolipids and sphingomyelin were separated by a silicic acid column, and finally purified by thin layer chromatography. Fatty acid, sphingosine and carbohydrate components of individual lipid classes were then analysed by gas-liquid chromatography. Nearly identical results were obtained with all viruses investigated. Approximately 20% of the total lipid was monohexosylceramide, distributed equally between glucosyl- and galactosyl- analogues. Lactosylceramide and oligohexosylceramides were found in much smaller concentrations (approx. 2%). About 15% of the total lipid was attributed to sphingomyelin. A large proportion of fatty acids (around 25% in sphingomyelin and 60% in glycolipids) belonged to the long chain (C19-C26) normal- and 2-hydroxy series. C18-sphingosine was found to be the only base present in all lipid classes investigated.


The effect of the phase transition on the hydration and electrical conductivity of phospholipids. Adsorption isotherms for various saturated phosphatidylcholines have been obtained. Lipids above and below their phase transition temperature differ only in the amount of water adsorbed and not in the nature of their adsorption isotherms. Cholesterol has an effect similar to that of increasing unsaturation in the hydrocarbon chains. Decreasing the length of the hydrocarbon chains for lipids below their phase transition temperature has no effect on the isotherms. If the chain length is short enough so that the lipids are above their transition temperature, however, a large increase in water adsorption occurs. All of the phospholipids exhibit a rapid increase of electrical conductivity for a few water molecules adsorbed per lipid molecule. All of the phospholipids show a saturation in conductivity at greater amounts of adsorbed water; the shape of the saturation region depends on whether the lipids are above or below their phase transition temperature. The activation energy for the electrical conductivity process depends on whether the hydrated lipids are in the "liquid-like" of the crystalline state, being lower for phospholipids in the liquid-like state. If the lipids are hydrated above their phase transition temperatures, their activation energies are lower than if they are hydrated below the transition temperature. Cholesterol lowers the activation energy. The phosphatidylcholines can be characterized by different activation energies, depending both upon their physical state and the presence of unsaturation in their hydrocarbon chains.


The phospholipid head-group orientation: effect on hydration and electrical conductivity. The water adsorption isotherms have been obtained for egg phosphatidyl-ethanolamine when it is complexed to egg phosphatidylcholine and cholesterol, respectively. In the presence of phosphatidylcholine, the phosphatidylethanol amine water binding is changed to a strong binding as compared to when the phospholipid is in its uncomplexed form. Cholesterol increases the water adorbed by the phospholipid, however, it does not change the nature of the isotherm. Phosphatidylmonomethylethanolamine also exhibits a strong water binding. The electrical conductivity of these phospholipids has been measured concurrently with their hydration. Electrical activation energies have been obtained for the fully hydrated phospholipids and are a function of both the amount of water adsorbed and the orientation of the polar head-group. The results are discussed in terms of a model for water adsorption, previously put forth by the authors.


Phospholipases A1 and A2 in bovine thyroid. In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.


Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors. A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.


Effects of local anaesthetics on the lipase of Rhizopus arrhizus. 1. A number of local anaesthetics were shown to inhibit the hydrolytic activity of a partially purified lipase from the mold Rhizopus arrhizur towards both triacylglycerol and phospholipid substrates. 2. Irrespective of whether triacylglycerol or phospholipid substrates were used, the inhibition was uncompetitive with respect to the substrate, independent of Ca2+ concentration, but pH dependent. 3. The inhibitory activity of the local anaesthetics studied closely paralleled the anaesthetic potency of the compounds.


Regulation of lipoprotein lipase. Induction by insulin. Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.


Isolation and structural studies of a sulfated sialosphingolipid from the sea urchin Echinocardium cordatum. Three sialosphingolipids have been isolated from a lipid extract of gonads of the sea urchin Echinocardium cordatum by partition dialysis and DEAE-cellulose column chromatography. The structure of the sialosphingolipid containing sulfate group has been established. On the basis of the results of total and partial acid hydrolysis, methanolysis, methylation, periodate oxidation and enzymatic hydrolysis with neuraminidase the sulfated sialosphingolipid was identified as 8-sulfate-sialyl-alpha-(2 leads to 6)glucopyranosyl-(1 leads to 1)ceramide. The long-chain bases were mainly phytosphingosine and its C16 homologue. The fatty acids of the sialosphingolipid were the mixture of normal and alpha-hydroxy fatty acids, their compositions were analysed by gas-liquid chromatography.


Isolation and partial characterization of human kidney gangliosides. 1. Eight gangliosides were purified from chloroform/methanol extracts of human kidneys by using modified Folch partition, dialysis, ethanol precipitation, silicic acid column chromatography and preparative thin-layer chromatography. 2. By thin-layer chromatographic behaviour and gas-liquid chromatographic determinations the main gangliosides in human kidney are N-acetylneuraminyllactosylceramide (74% of total) and di-N-acetylneuraminyllactosylceramide (19% of total). 3. Five hexosamine-containing fractions were isolated. Four of them were homogeneous on thin-layer chromatography, and one contained two gangliosides. By gas-liquid chromatography-mass spectrometry it was shown that two gangliosides (together 5% of total) contain glucosamine, and one (1% of total) contains galactosamine. The other of the glucosamine gangliosides contains fucose in addition to the usual sugars found in gangliosides. Of the two remaining hexosamine positive fractions (together 1% of total) one was homogeneous on thin-layer chromatography, the other contained two gangliosides. These two fractions contained both glucosamine and galactosamine. 4. The main long-chain base in all fractions was sphingosine.


The isomerization of 2,5- and 9,12-octadecadienoic acids by an extract of Butyrivibrio fibrisolvens. A cell-free particulate preparation from Butyrivibrio fibrisolvens was used to study the relative rates of isomerization of all cis,cis-methylene-interrupted isomers of octadecadienoic acid. Only two isomers were found to be substrates, the 9,12-isomer was isomerized at 41 +/- 4 mumol/min per mg protein, and the 2,5-isomer at 11 +/- 1 mumol/min per mg. The product of the isomerization of the 2,5-isomer had an ultraviolet absorption maximum at 233 nm indicating that it was the 3,5-isomer. The isomerization of the 2,5-isomer was studied in detail. Its rate of isomerization was linear with protein concentration up to 0.047 mg/ml, and was linear with substrate concentration up to 48 muM. The pH optimum was 6.8. Below pH 6, the substrate was also subject to spontaneous isomerization. The inhibition of isomerization of the 9,12-isomer by the other isomers was studied. Those isomers in which the double bonds are close to the carboxyl group were the most effective inhibitors. The preparation was also found capable of hydrogenating the conjugated diene product from the 2,5-isomer to a monoene after prolonged incubation.


Metabolism of [14C]arachidonic acid by human platelets. A time dependent incorporation of [1-14C] arachidonic acid into platelet phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine was observed in platelet-rich plasma. When platelets, so labelled, were washed and treated with thrombin, there was a major decrease in the radioactivity of phosphatidylcholine and phosphatidylinositol. This decrease was accounted for by the appearance of several previously identified (Hamberg and Samuelsson (1974) Proc. Natl. Acad. Sci. U.S. 71, 3400) 14C-labelled oxygenated products of arachidonic acid.


A chemical and physical method for determining the complete base composition of plant DNA. Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.


Intraction of aminoacyl-tRNA synthetases with ribosomes and ribosomal subunits. Salt-washed ribosomes from rabbit reticulocytes stimulate seven partially purified aminoacyl-tRNA synthetases up to threefold: arginyl-, alanyl-, isoleucyl-, lysyl-, methionyl-, phenylalanyl- and valyl-tRNA synthetase. Both isolated subunits but not total ribosomal RNA show the stimulation. The increase in synthetase activity is also found in a highly purified Escherichia coli system employing homogenous (E. coli)phenyl-alanyl-tRNA synthetase and (E. coli)- tRNAPhe. The biological significance of the stimulation is discussed.


Translation of Rauscher  leukemia virus RNA in heterologous cell-free systems. Rauscher leukemia virus RNA (RLV RNA) is translated in mammalian cell-free systems into distinct polypeptides which are immunoprecipitable by an antiserum directed against RLV proteins. These polypeptides partially comigrate electrophoretically with native viral proteins synthesized in vivo in JLS-V9 cells. Besides 72000-, 65000- and 50000-dalton polypeptides a 15000-dalton polypeptide is also synthesized in vitro. Analysis of incubations of RLV RNA in different cell-free systems reveals that no virus-specific factors are required in the translation of RLV RNA in vitro.