Personal questionnaire changes and their correlates in a psychotherapeutic group. Personal questionnaires were constructed for seven members of a psychotherapy group. Self-reported changes during group sessions and over a period of up to 10 months' treatment were analysed. There was no general pattern of 'improvement' either during sessions or in the longer term. There was evidence suggestive of an inverse relationship between changes during sessions and over the period of the study. Some clinically meaningful relationships were discerned between the present data and repertory grid and verbal behaviour measures on the same group members reported in a previous paper. Theoretical and methodological implications are discussed.


Some correlates of repertory grid measures used to study a psychotherapeutic group. Repertory grid data provided monthly by the members of a psychotherapeutic group were related to measures of verbal behaviour during group sessions in ways which were both statistically significant and psychologically meaningful. There was evidence that the group members re-enacted earlier patterns of family relationship in their mutual interaction. For individual patient members of the group, speaking, being spoken to, and introducing several kinds of topic into the group discussion had significant associations with grid variables implicating self-esteem and patterns of identification with parents; but the correlation patterns varied between patients.


The family and the problem of internalization. This paper is an attempt to integrate concepts derived from family therapy with psychoanalytic concepts. In particular, the nature and process of internalization is scrutinized in the light of family-role functioning.


The effect of children upon the marriage relationship. A marital questionnaire, completed by 40 fertile and 50 infertile couples, revealed greater unanimity of response between childless partners, who may however have been tempted to idealize their relationship. It is suggested that parenthood may limit the scope of communication between spouses. Childlessness may be stressful but it can also make for closer mutual affinity in the couple's thoughts and feelings.


Neonatal mortality of breech deliveries with and without forceps to the aftercoming head. A retrospective study of 1423 liveborn breech deliveries occurring in one hospital from 1964 to 1973 inclusive showed that application of forceps to the aftercoming head of infants between 1 and 3 kg birth weight was associated with a significant reduction in neonatal mortality.


The distribution of placental lactogen in the human term placenta. Serial frozen sections of human term placentae were examined by quantitative morphology and, after extraction, by a radioimmunoassay for human placental lactogen (HPL). The total quantity of HPL in the placenta was estimated as 720 mg. The amount of HPL was related to the amount of trophoblast in any area, but otherwise did not vary in different parts of the placenta. A semi-logarithmic relationship was observed between the amount of HPL and the percentage trophoblast in a section. It is suggested that the amount of HPL in a given area of the placenta is related to the blood-flow in that area.


Plasma oestradiol and progesterone estimation for the monitoring of induction of ovulation with clomiphene and chorionic gonadotrophin. Two hundred and thirty-five courses of treatment with clomiphene and human chorionic gonadotrophin (HCG) were administered for the induction of ovulation in 75 infertile women (three with primary amenorrhoea, 45 with secondary amenorrhoea and 27 with oligomenorrhoea) of whom 61 had previously been unsuccessfully treated with clomiphene alone. The treatment was monitored by plasma oestradiol estimations on day 15 and day 22 in 103 cycles and by plasma progesterone estimations on day 22 in all 235 cycles. Injections of HCG were given on day 15 and 22. Twenty patients failed to respond to treatment (43 courses). The remaining 55 patients showed no response in 32 courses, deficient responses in 55 cycles and adequate responses in 105 cycles (plasma progesterone level exceeding 10 ng per ml on day 22). The mean values (+/- SD) of plasma oestradiol on day 15, reflecting follicular development, were: 73 +/- 30 pg per ml for cycles with no response; 255 +/- 182 for deficient cycles and 366 +/- 226 pg per ml for adequate cycles. There was a positive correlation (r = 0-57) between day 15 plasma oestradiol and day 22 plasma progesterone values. Increased clomiphene dosage resulted in higher plasma oestradiol and progesterone levels. Thirty-nine pregnancies occurred in 34 out of 55 patients capable of response (62 per cent). The plasma progesterone levels during conception cycles were higher than those in non-conception cycles in the same patients. Monitoring of induction of ovulation by plasma hormone estimations allows detection and correction of inadequate response.


A study of the secretory immune system of the female genital tract. Immunocytes in the female genital tract are found principally in the endocervix. The predominant immunoglobulin produced in this site is IgA and this, together with the presence of secretory component in endocervical epithelium, indicates that a local secretory immune system, similar to that found in the gastrointestinal and respiratory tracts, exists in the endocervix. This local secretory immune system appears to be the major source of immunoglobulins in cervical mucus and the presence of secretory IgA in cervico-vaginal secretions provides an effective protection mechanism against viral and bacterial infections. Secretory IgA also acts non-specifically against exogenous antigens and may, by binding spermatozoal antigens, play a role in immunologically mediated infertility.


An abdominal approach to the repair of post-hysterectomy vaginal inversion. An abdominal operation is described using a Mersilene mesh bridge for the correction of post-hysterectomy vaginal inversion. Four cases are reported in which a satisfactory anatomical and functionally effective repair has been achieved.


Postpartum renal failure associated with eclampsia and penicillin hypersensitivity. A case of acute renal failure associated with disseminated intravascular coagulation occurring immediately post partum is reported. The aetiological role of the associated eclampsia together with evidence for a hypersensitivity reaction to ampicillin is discussed.


The diagnosis of early pregnancy failure by sonar. In a series of 425 consecutive patients examined by sonar in the first half of pregnancy 176 ultimately aborted. On analysis of the sonar and post-abortion findings it was found that the aborted pregnancies fell into five clearly defined groups; blighted ova or anembryonic pregnancies, missed abortions, hydatidiform moles and early and late live abortions. The blighted ova and the missed abortions comprised by far the largest and the early live abortions the smallest groups. Strict diagnostic sonar criteria of abnormality, independent of menstrual or clinical histories, were established for the first three of the groups, and an absolute diagnosis could be made at the time of the first examination in all cases of missed abortion and hydatidiform mole and in just over half of the cases of blighted ovum, the remainder requiring a second and occasionally a third examination. In the first half of the study the majority of the patients were allowed to abort spontaneously but with increasing confidence in the techniques patients were offered termination whenever the diagnosis of an abortive pregnancy was made. Anticipation of fetal death in utero or impending abortion of a live fetus proved to be a much more difficult problem, and in only those patients who aborted a live fetus before the tenth week of pregnancy did the sonar examination reveal any significant abnormality. Possible aetiological backgrounds to these groups of abortions are discussed in the light of the sonar findings.


Prognosis in threatened abortion: a comparison between predictions made by sonar urinary hormone assays and clinical judgement. One hundred patients admitted to hospital with a diagnosis of threatened abortion were assessed by means of sonar, urinary oestrogen, pregnanediol and human chorionic gonadotrophin (HCG) assays and clinical examination. Assay of oestrogen excretion was the most accurate (86-5 per cent) in predicting the ultimate outcome of pregnancy, but did not give as much information as sonar examination which gave an accurate prognosis in 84 per cent of cases and was much quicker to perform The reasons for the sonar failures are discussed. Assay of urinary pregnanediol excretion gave an accurate indication of outcome in 74 per cent of cases and 24-hour urinary HCG in 70 per cent although random urinary HCG estimations provided an accurate prediciton in only 54-5 per cent of cases. Clinical examination presented the usual difficulties in assessing uterine size and predicting abortion from the amount of bleeding and pain.


Factors affecting the increasing incidence of severe non-haemolytic neonatal jaundice. Neonatal hyperbilirubinaemia is increasing in frequency. In view of conflicting evidence about the possible causes, retrospective analyses have been carried out among babies born during six months of 1974. Preliminary analysis confirmed the over-riding importance of preterm birth (before 37 weeks), but only one of 17 such cases could be attributed to ill-judged artificial induction of labor. For the main analysis, the incidence of eight possibly relevant antecedent factors was compared in 46 cases of hyperbilirubinaemia (unconjugated bilirubin more than 15 mg per 100 ml in term babies and more than 13 mg per 100 ml in some preterm babies) and in 92 controls matched for sex and gestational age. Induction of labour by "primary" oxytocin infusion and artificial rupture of the membranes was very significantly more common in the index cases (p less than 0-01), but there was no difference in the incidence of "secondary" oxytocin, used to accelerate spontaneous labour. Evidence of uterin unresponsiveness suggests that the natural onset of labour was being anticipated by at least some days in many of the index cases and this could prevent the natural "priming" of the fetal enzyme systems. An excess of epidural analgesia in the mothers of the index cases was probably due to its association with the need for pain relief during "primary" oxytocin infusions. The higher incidence of postnatal weight loss in the index cases presumably contributed to the hyperbilirubinaemia.


Cigarette smoking in pregnancy: differences in peripheral circulation between smokers and non-smokers. Ten smokers and ten non-smokers were followed serially from 12 to 16 weeks of pregnancy to six weeks postnatally. Measurements of calf and foot blood flow and toe and finger skin temperature were made at 4 to 6 week intervals in both groups, and the acute effects on the peripheral circulation of smoking one standard cigarette were also measured in the smokers at each stage of pregnancy. Comparison between the smokers and non-smokers showed that in the last trimester of pregnancy smokers had significantly higher foot blood flow and toe and finger temperature than non-smokers. These circulatory variables rose during the period of pregnancy studied in smokers but altered little in non-smokers. There was no significant difference in calf blood flow between the groups. Smoking a cigarette during pregnancy caused a fall in foot blood flow and in finger and toe skin temperature. Smoking increased calf blood flow between 12 and 25 weeks of pregnancy but decreased it in late pregnancy. Previous investigations of blood flow in pregnancy have not differentiated between smokers and non-smokers. It is suggested that the rise in foot blood flow and skin temperature believed to occur generally during pregnancy applies mainly to smokers. Some possible reason for differences in skin blood flow between smokers and non-smokers in pregnancy are discussed.


Plasma unconjugated 15alpha-hydroxyoestriol (oestetrol) levels in normal human pregnancy. Serial measurements were made of the concentration of plasma unconjugated oestetrol in normal human pregnancy. The mean curve and limits of normal variation have been established. Plasma levels rose from just below 2 ng/ml at 20 weeks to approximately 5 ng/ml at term.


Metabolism of prostaglandins within the pregnant uterus. The metabolism of E and F prostaglandins has been studied in both fetal and maternal tissues obtained from the pregnant human uterus. Both 15-hydroxyprostaglandin dehydrogenase and 13,14-prostaglandin reductase are shown to be widely distributed in these tissues. Comparison of the levels of enzyme activity in different tissues demonstrates that the greatest metabolism of prostaglandins takes place in the membranes and placenta and that there is much less in the myometrium and the decidua. The lowest enzyme activity was found in the umbilical cord, and no metabolism could be demonstrated in amniotic fluid or in umbilical venous blood. The significance of these findings is discussed.


The routine use of oral prostaglandin E2 in induction of labour. Oral PGE2 when combined with amniotomy was found to be safe and also an effective agent for induction of labour at term even when intensive monitoring is not possible for all patients as is the case in the University Department in Kandang Kerbau Hosital which has 9000 deliveries per year. It was found to be most useful in patients with a pelvic score of 5 and above and especially in multigravidae. It obviates the discomform and difficulties in maintaining an intravenous infusion.


Serial human placental lactogen estimations in serum and placental weight-for-dates. The relation between serial HPL assays in serum and placental weight-for-dates was studied in 70 randomly chosen pregnant women. Out of five different aspects of the HPL curve only a fall below the 2-3d centile without subsequent recovery was related to low placental weight. When a small-for-dates (SFD) placenta was associated with normal HPL levels, maternal body weight tended to be lower than if both placental weight and HPL levels were abnormal. This suggests that physiologically small placentae are discernible from pathologically small placentae by a normal HPL curve.


Pregnancy in the overweight Nigerian.

Pregnancy in the overweight Nigerian. One hundred pregnant patients weighing 80 kg or over at the 14th week of pregnancy were studied during their antenatal period and delivery. The incidence of pre-eclampsia, hypertension, pyelitis of pregnancy and operative delivery was higher in overweight patients than in an equivalent number of patients of average weight also studied. The age-related difference in antenatal weight gain in obese patients was confirmed. Two maternal deaths occurred among the overweight patients.


Maternal serum sialomucins during pregnancy and postpartum in patients with pre-eclampsia. The seromucoid fraction of maternal serum glycoproteins is increased in pre-eclampsia and the rise, which is proportional to the severity of the disease, may reflect progressive placental deterioration with increasing trophoblast fragmentation and deportation. The pulmonary lysis of deported trophoblast effects the local release of fetal antigens and thromboplastin in the lungs. The former, which are immunologically inert because they are complexed with maternal serum seromucoid, are released into the peripheral circulation, while the local coagulation hazard presented by the latter is countered by intensified fibrinolytic activity, the products of which are also released into the peripheral circulation. In severe pre-eclampsia the peripheral circulation contains a considerable excess of physically similar proteins derived from the seromucoid fraction and fibrin degradation products, and the conditions which then obtain in the renal glomerulus favour the physical process of coacervation. In that event the mechanism of the origin and development of the renal lesion can be described in purely physical terms.


Vaginal candidiasis and the role of the digestive tract as a source of infection. Of 300 female patients seen in a Venereal Diseases clinic, 84 (28-0 per cent) were found to be harbouring Candida albicans or C. parapsilosis in the genital tract and 33 per cent of these patients had clinical signs of vulvovaginitis. Of the 84 patients, 71-4 per cent harboured yeasts in the ano-rectal tract; of the remaining 216 patients, 25-9 per cent harboured yeasts in the ano-rectal tract. There was no evidence to suggest that the incidence of yeasts in the mouth, ano-rectal or genital tracts changed with age or use of oral contraception.


Summary code for ocular herpes simplex.

Summary code for ocular herpes simplex. A code for herpetic eye disease is presented. This simplifies the management of individual patients, and the up-to-date code summary of each case is easily maintained thus facilitating assessment of interrelating factors. It is suggested that the pattern of herpetic eye disease may have prognostic and epidemiological significance. This system has simplified retrieval of such data.


Use of random-dot sterograms in the clinical assessment of strabismic patients. Random-dot stereograms were shown to a sample of strabismic patients for whom there was clinical evidence of stereopsis. Two kinds of sterograms were used, one of the usual sort and the other having a contour surrounding the disparate area in each field of view. The patients tested could be clearly classified as belonging to one of three response groups. The first group could fuse both kinds of stereogram, the second could fuse only he contoured kind, and the third could not fuse either kind. This grouping was found to relate to the degree to which bifoveal binocular single vision had been absent in the clinical histories of these patients. The result is discussed in terms of the consequences for vergence and stereopsis of period of absence of normal binocular function.


Graduated recession of the superior oblique muscle. Recession of the superior oblique was performed bilaterally in 12 patients with the A phenomenon and unilaterally in four patients with vertical imbalance. The results are discussed.


Electrovitreotomy. 2. Principles and results. Continued experience confirms the suitability of electrovitreotomy for dividing pathogenically taut proliferative and non-proliferative vitreous forms with a minimum of bleeding, and an inability to cut vitreous of ordinary consistency and tension. It differs directly with electrovitrectomy in these regards. To use effectively with a minimum of complications it is mandatory to have a vitrectomy capability immediately at hand and to respect a wide variety of interrelated factors that include: current density, electrical resistance of tissues, duration of electrical action: dimension, insulation, and configuration of the electrode: configuration, tension, location, and morphological content of dissectable structures. Mobile and dispersed blood in the liquid of the retrovitreal space often surrounds cuttable vitreous lesions. Frequently rest causes it to sediment and improves viewing sufficiently to permit effective electrovitreotomy without vitrectomy.


Corneal rust removal by electric drill. Clinical trial by comparison with manual removal. The dental burr rotated by an electric drill is the quickest, safest, and most precise form of treatment for corneal rust rings. It enables complete removal of the corneal rust at a single treatment and leaves a smooth crater that is no larger than the original rust ring. Pain relief is more rapid after electric drill removal; this is probably related to the complete removal of the rust. Epithelial and stromal healing are marginally faster than after manual removal and the patients'  duration of attendance is less. The ideal drill is a slim straight instrument, which rotates dental burrs and is operated by a light finger pressure. A brake which stops drill rotation on lifting the finger is a useful safety feature.


Identification of copper ions in aqueous and vitreous of eyes containing copper and iron foreign bodies. The reliability of aqueous analysis by the atomic absorption spectrophotometer for copper ion content in samples from eyes containing intravitreal pure copper particles was investigated. It was demonstrated that values of copper ions rose well above the normal in the aqueous as well as in the vitreous of such eyes. The standard deviations of the mean values were generally high. It was also found that a similar copper ion content increase occurred in eyes containing iron particles. The possible clinical implications of these findings are discussed.


Trachoma in the Sudan. An epidemiological study. The prevalence and distribution of trachoma in the Sudan has been studied. The morbidity rate of 83.2 per 1000 in the Northern Province decreases southwards until it reaches 0.94 in the extreme south (Equatoria Province). 2. In endemic areas infection starts very early--in the first year of life. 3. There is a marked difference between the prevalence of trachoma in towns and villages--71.3 per cent for the age group 1-4 years in villages, and 56.7 per cent for the same age group in the towns. This is probably due to better standards of living and hygiene in the towns. 4. Some relationship has been found between the rainfall, the relative humidity, and the incidence of trachoma, but this does not necessarily apply in other countries. 5. The factors that may explain the high prevalence of trachoma in the northern Sudan are: a. Mechanical trauma caused by frequent sandstorms. b. Irritation of the eyes by dust particles, leading to excessive watering and discharge, and rubbing with the fingers. c. The habit of frequent hand-shaking. d. Poor personal hygiene in pre-schoolchildren. e. Associated bacterial conjunctivitis. f. The presence of eye-seeking flies.


Prevalence of diabetic retinopathy. Age, sex, and duration of diabetes. From a list of all patients seen at the Joslin Clinic in a recent 6-month period, a random sample of 965 was selected for record review by a clinic assistant. A house physician specializing in diabetes then verified and adjusted the assistant's work as necessary. The data were analyzed both by simple cross-classification and by fitting a multiple logistic risk function. As often reported previously, we found a strong positive association between retinopathy and duration of diabetes. One of our findings which has not been so clearly reported in earlier papers is that the positive association between retinopathy and age is limited to the group with diabetes of less than 10 years' duration. We also found sex differences in retinopathy prevalence which were not large enough to rule out attributing them to chance.


Atypical cotton-wool spots.

Atypical cotton-wool spots. Atypical cotton-wool spots in four selected cases of diabetic retinopathy are discussed. The most striking characteristic distinguishing them from typical cotton-wool spots is their size, which ranges from about 2 to 4 disc diameters. They develop after stenosis or a complete obstruction of a first order arteriole at the point at which it emerges from the parent arterial branch. The size of the lesion corresponds with the size of the affected arteriole. Restoration of local circulation is attempted by neighbouring arterioles and venules, the latter carrying a reversed blood flow. Arterio-venous communications along the border of the infarcted area are a prominent feature, most likely attributable to the necessity of free drainage of the arterial influx. The same haemodynamic principle applies to the coarsening of the adjacent capillary bed. Secondary changes of the bordering venous branches include aneurysm formation and the staining of the venous wall.


Retinal macroaneurysms.

Retinal macroaneurysms. Twenty patients with retinal macroaneurysms were examined and observed for at least 9 months. All these patients were elderly and had evidence of hypertension or systemic vascular disease. Nineteen patients presented with loss of central vision and one patient was asymptomatic. Visual loss was due to macular haemorrhage in 13 patients and macular oedema in 6. Whereas the visual prognosis in patients with haemorrhage was good it was considerably poorer in the patients with macular oedema.


Genetic analysis of cover test measures and AC/A ratio in human populations with varying incidences of strabismus. The genetic analyses of indices relating to distance alignment and accommodative convergence are presented. This approach is important in understanding the contribution of genetic factors to observed individual differences for these measures. Abnormalities of either of these two components results in a tendency towards either a convergent or divergent position of the eyes (relative to the position of fusional demand) and thus places an additional load or strain on the other components of the binocular mechanism which must compensate for this potential disruption. If compensation is inadequate to maintain alignment of the eyes, a manifest deviation will result. Consequently, an understanding of the aetiology of such factors underlies an understanding of the aetiology of nonparalytic strabismus. The three populations displayed different tendencies in the cover test measure. The average tendency for each of the populations was consistent with the type of deviation common to all propositi of families within each of the respective populations. The heritability of this character was similar in the three populations and was dependent on the contribution of the female parent only, but to a substantial degree (h2 = 0-42 +/- 0-12). The nature of the sex difference is at present open to speculation. The population means within generations were similar for the gradient measure of AC/A ratio. The heritability of this character (0-38 +/- 0-09) suggests that the genetic component is of substantial importance underlying individual differences for AC/A ratio.


Superior oblique tendon sheath syndrome. Clinical examination of 15 patients with the superior oblique tendon sheath syndrome showed that 12 of them had some evidence of stenosing tenosynovitis.


Ross' syndrome (tonic pupil plus).

Ross' syndrome (tonic pupil plus). Two cases of tonic pupil, hyporeflexia, and segmental hypohidrosis (Ross' syndrome) are reported. The relationship of this syndrome to other autonomic dysfunction is discussed. Those symptoms (emotional instability, loss of sweating, orthostatic hypotensive symptoms, and signs of bilaterality of the tonic pupil) which should alert the clinician to more extensive disease states are noted. It is suggested that these conditions may represent a continuum or spectrum of disorders with a widespread degree of severity and progression.


A new approach to the study of phospholipid-protein interactions in biological membranes. Synthesis of fatty acids and phospholipids containing photosensitive groups. In a general approach to the study, in vivo and in vitro, of the interactions between phospholipids and proteins in biological membranes, a variety of fatty acids containing photosensitive groups in different positions in the alkyl chains has been synthesized. The fatty acids synthesized include: 16-azidopalmitelaidic acid, 12-azidooleic acid, 6-, 9-, 11-, and 12-azidostearic acid, 12-omicron-(ethyl-2-diazomalonyl)stearic and -oleic acids, 12-omicron-(4-azido-2-nitrophenyl)stearic and -oleic acids, and 12-oxo-10-octadecenoic acid. Some of the above synthetic fatty acids were also prepared in the radioactively labeled form. For in vitro studies, many of the above fatty acids were used to acylate the 2 position in the preparation of a number of mixed acylphosphatidylcholines and mixed acylphosphatidylethanolamines. On sonication, the synthetic phospholipids formed sealed vesicles. Intermolecular cross-linking of the fatty acyl chains in phospholipids was demonstrated on photolysis of the vesicles.


The subunit structure of thymus leukemia antigens. EDTA-containing buffer solubilizes thymus leukemia antigens (TLa) from crude thymocyte membrane fractions. The TL antigens consist mainly of molecules of a size similar to immunoglobulin G when gel chromatography analyses were performed under physiological conditions. A single component of TLa was apparent on sucrose density gradient ultracentrifugation of solubilized thymocyte membrane macromolecules as monitored by indirect immunoprecipitation. The sedimentation constant for the TL antigens (5.8 S) was considerably less than that for immunoglobulin G. The gel chromatography and ultracentrifugation data suggest an apparent molecular weight for TLa of about 120000. TLa isolated by indirect immunoprecipitation is composed of two types of polypeptide chains. The smaller subunit was identified as beta2-microglobulin. The larger polypeptide chain carried the alloantigenic determinants and displayed a molecular weight of about 50000 after reduction and alkylation. TLa subjected to molecular weight determination under denaturing conditions was composed of two components. The smaller component was beta2-microglobulin which evidently is linked to the larger polypeptide chain by noncovalent interactions only. The larger component had a size greater than reduced and alkylated immunoglobulin G heavy chains. Upon reduction and alkylation of the latter component its size was reduced and it appeared to have a molecular weight of about 50000. Consequently, TLa is composed of two disulfide linked heavy polypeptide chains and two beta2-microglobulin molecules. TLa solubilized by papain digestion comprises two polypeptide chains, one of which is beta2-microglobulin. The larger 37000-dalton subunit is a fragment of the heavy polypeptide chain. This was demonstrated by digesting solubilized 120000-dalton TLa with papain. The proteolytic fragments obtained were indistinguishable from those directly released from the cell surface by proteolysis. The papain-derived TLa fragment exhibited most if not all the alloantigenic determinants.


Studies on the Methyl Green-DNA complex and its dissociation by drugs. Spectrophotometric results indicated that Methyl Green bound stably to native calf thymus DNA and to poly[d(A-T)] but to a lesser extent to phiX 174 DNA, tRNAs, and poly(dG-dC), a copolymer that exists preferentially in the A conformation. Exposing the Methyl Green-DNA complex to graded concentrations of ethyl alcohol liberated part of the dye slowly by a zero-order reaction; higher alcohol concentrations which cause the B leads to A transition of DNA released the bulk of Methyl Green. The viscosity of the Methyl Green-DNA complex was significantly lower than that of the uncomplexed DNA. The dye was progressively liberated from DNA by 1.5 x 10(-1) M NaCl and by much lower concentrations of Mg2+; in its stoichiometric complex with DNA, it increased Tm by approximately 12 degrees C. A series of DNA-complexing drugs displaced Methyl Green from DNA at exponential rates and to end points which were correlated. End points of displacement correlated with the abilities of drugs to unwind supercoiled DNA, to labilize ribosomes to heat, and to eliminate a kanamycin resistance determinant from an R factor carried by Salmonella typhimurium. Additional correlations between Methyl Green displacement and biochemical-biological activities of displacing drugs are cited. In conjunction with these findings, our results suggest that Methyl Green displacement analysis is a useful biochemical screen for the detection or development of biologically active compounds which bind to DNA.


Purification of human placenta phenylalanine, valine, methionine, glucine, and serine transfer ribonucleic acids. By using column chromatography on varied media, the purification of several individual tRNAs from human placenta has been achieved. The crude human placenta tRNA was isolated using phenol extraction at pH 4.5 followed by DEAE-cellulose chromatography (B. Roe (1975), Nucleic Acids Res. 2, 21-42) and initially fractionated on BD-cellulose at neutral pH. Subsequent chromatography of the partially purified tRNA using high-speed, high-pressure liquid chromatography on RPC-5 and Aminex A-28 coupled with chromatography on BD-cellulose at acidic pH and on DEAE-Sephadex A-50 significantly shortened isolation time for milligram quantities of several pure tRNA species. Those tRNAs from human placenta obtained in a purity greater than 1.2 nmol/A260 unit are tRNAPhe, tRNAMet(i), tRNAVal(1a), tRNAVal(1b), and tRNAGly(1), while those obtained at purity of at least 0.8 nmol/A260 unit are tRNASer2 and tRNASer3. In addition, the use of Aminex A-28 as a chromatographic system for the isolation of tRNA is discussed.


Alkyl isocyanates as active site-specific reagents for serine proteases. Location of alkyl binding site in chymotrypsin by X-ray diffraction. The structure of octylcarbamoyl-alpha-chymotrypsin to a resolution of 3.0 A is described. The n-octyl side chain of the active site directed irreversible inactivator octyl isocyanate is bound exclusively in the hydrophobic substrate binding pocket. The n-octyl isocyanate forms a planar urethane bond with the Ser-195 Ogamma and extends approximately 1 A deeper into the hydrophobic pocket than the indolyl group of indoleacryloyl-alpha-chymotrypsin (Henderson, R. (1970), J. Mol. Biol. 54, 341). All the structural changes are essentially identical with those observed in indoleacryloyl-alpha-chymotrypsin including the observation of a hydrogen bonded water molecule between the carbonyl oxygen of the octylcarbamoyl group and the imidazole group of His-57. The observed mode of n-octyl alkyl binding to chymotrypsin is consistent with the hypothesis proposed earlier (Brown, W. E. and Wold, F. (1973), Biochemistry 12, 828).


Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin. The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.


The linkage between oxygenation and subunit dissociation in human hemoglobin. Consequences for the analysis of oxygenation curves. For human hemoglobin, a pronounced dependence of oxygenation curves upon protein concentration can be demonstrated experimentally in the range between 10(-4) and 2 X 10(-6) M heme. The effects of such protein concentration dependence upon analysis of saturation curves have been explored using a model-independent linkage analysis which incorporates the dissociation of tetramers to dimers. We have carried out stimulations of oxygenation curves representing a variety of energy distributions designed to cover a wide range of values which are relevant to known hemoglobin systems and experimental conditions. The resulting simulated oxygenation curves were analyzed by least-squares minimization procedures in terms of the tetramer binding isotherm to yield the four apparent Adair constants. These derived constants were compared with the originally assumed values used in the simulation in order to assess the extent to which their values may be altered by the presence of dimer. For each energy distribution the analysis has been carried out over a wide range of protein concentration. We have found that the presence of even small amounts of dimer that are necessarily present at the low protein concentrations commonly employed may have a devastating effect upon the reliability of Adair constant determinations. In addition to these simulated cases, we have analyzed two sets of highly precise experimental data from the literature in order to assess the degree to which constants obtained may have been influenced by the presence of dimer.


Molecular mobility and structure of elastin deduced from the solvent and temperature dependence of 13C magnetic resonance relaxation data. 13C relaxation parameters, T1, line width, and NOE, have been determined for backbone carbons of ligamentum nuchae elastin swollen by 0.15 M NaCl, 0.15 M NaCl-formamide, 0.15 M NaCl-ethanol, dimethyl sulfoxide, and formamide. The data have been analyzed in terms of (a) a single correlation time model and (b) a model employing a log-chi2 distribution of correlation times used by Schaefer (1973) to analyze solid cis-polyisoprene 13C relaxation data. Employing the latter mode, one obtains an approximately self-consistent quantitative analysis of all the elastin data. An average backbone correlation time, tau, of ca. 2 nsec is calculated for elastin swollen in the presence of polar organic solvents at 37 degrees, in approximate agreement with tau of 0.4 nsec obtained for bulk cis-polyisoprene at 35 degrees. The influence of solvent and temperature on elastin spectra indicate that the larger tau value (approximately 80 nsec) obtained for elastin swollen by 0.15 M NaCl at 37 degrees is a consequence of weak interchain polar and hydrophobic interactions, a result which is in accord with the reported viscoelastic behavior exhibited by water-swollen elastin at 37 degrees. The results obtained further suggest that Gly, Pro, and Val residues are significantly more mobile than Ala residues, which are located in the cross-link regions. Hence, the NMR data support the view that water-swollen elastin is composed of a network of mobile chains, except possibly in the cross-link regions.


Partial purification of "omega" protein from calf thymus. Proteins which relax supercoiled DNA, called "omega" proteins, are thought to be involved in DNA replication. Calf thymus is a plentiful source of "omega" protein activity. It has been extensively purified and has been characterized as behaving similarly to other eukaryotic "omega" proteins in completely relaxing either positively or negatively supercoiled DNA, requiring a salt concentration of about 0.2 M NaCl or KCl, and not requiring Mg2+. A transient nick must occur but could not be detected. A new assay for "omega" activity is described which is rapid and sensitive, and depends on the fluorescence enhancement of ethidium intercalating duplex DNA.


Chemical synthesis of N beta-oxalyl-L-alpha, beta-diaminopropionic acid and optical specificity in its neurotoxic action. A practical procedure is described for the bulk synthesis of the neurotoxin N beta-oxalyl-L-alpha, beta-diaminopropionic acid (OA2pr3), a potential dicarboxylic amino acid antagonist of Lathyrus sativus seeds. L-Aspartic acid was reacted with sodium azide in 30% fuming sulfuric acid and L-alpha, beta-diaminopropionic acid hydrochloride (A2pr3-HCl) was isolated in yields greater than 75%. Potassium methyl oxalate was found to react selectively with the beta-amino group of S2pr3 resulting in near quantitative yields of OA2pr3. D-OA2pr3 has been made for the first time by this procedure. Unlike L-OA2pr3 the naturally occurring neurotoxin, D-OA2pr3, is not neuroactive even in high doses. The microsynthesis of L-[2,3-3H]A2pr3 from L-[2,3-3H]aspartic acid is also described, and the same procedure could also be used to prepare the neurotoxin with other labels. The availability of the neurotoxin in bulk and in labeled form should further experimental approaches to the understanding of its mechanism of action.


Substrate specificity of ribosomal peptidyltransferase. Effect of modification in the heterocyclic, carbohydrate and amino acid moiety of 2'(3')-O-L-phenylalanyladenosine. The chemical synthesis of 2'(3')-O-L-phenylalanyl derivatives of nebularine (Ld), 6-methoxynebularine (Ih), N6,N6-dimethyladenosine (Lk), 6-methylthionebularine (Lo), 8-bromoadenosine (Lr), tubercidin (Iu), and 3'-O-L-phenylalanyl derivatives of 1-(beta-D-arabinofuranosyl)cytosine (IIIg) and 9-(beta-D-arabinofuranosyl)adenine (IIIl) is described. 2'(3')-O-(3-Phenyl)propionyladenosine (Iv) was obtained by reaction of adenosine with ethyl 3-phenylorthopropionate and subsequent hydrolysis of the orthoester intermediate IV with formic acid. Compounds Id, Ih, Ik, Io, and Iu were active in the release of Ac-Phe from N-Ac-Phe-tRNA-poly(U)-70S ribosome complex: at 0.01 mM the release of Ac-Phe was 50-100% of that of A-Phe. At 1 mM, compounds Ir and IIIg released 30 and 25% of Ac-Phe relative to A-Phe whereas derivatives Iv and IIIl were virtually inactive. The results indicate the following conclusions regarding ribosomal peptidyltransferase activity of 2'(3')-O-aminoacyl nucleosides: (a) the presence of the 2'-hydroxy group in the ribo configuration is more important for a highly active substrate (A-Phe) than for one of moderate activity (C-Phe); (b) the heterocyclic (purine) residue is in the anti conformation although this requirement is not absolute; (c) the presence of the amino group of the aminoacyl moiety is required; (d) acceptor activity is dependent upon the substituent in the position 6 of the purine moiety.


Gas chromatography-mass spectrometry for probing the structure and mechanism of action of enzyme active sites. The role of Glu-270 in carboxypeptidase A. A new technique for the study of the mechanism of enzymes has been developed. An enzyme, modified by an active-site directed reagent, is digested by one or more proteases. The resulting mixture of oligopeptides is then analyzed directly by gas chromatography-mass spectrometry without the use of separation or isolation procedures. A comparison with unmodified enzyme identifies the modified residue as well as quantifies the reaction. This approach has been applied to the identification of Glu-270 in the active site of carboxypeptidase A using a carbodiimide as modification reagent. Studies on the possible incorporation of 18O (from 18O-enriched water) into Glu-270 or other acidic residues near the active site of carboxypeptidase A show that the oxygens of the carboxyl groups of these residues are not exchangeable.


A cell free system from HeLa cells active in initiation of protein synthesis. A cell free system programmed by endogenous mRNA and active in initiation of protein synthesis has been obtained from HeLa cells by adding 25-100 muM hemin to the medium used to homogenize the cells. Hemin stabilizes the initiation activity of the extract, which otherwise decays rapidly even at 0 degrees C. The role of hemin in promoting initiation has been examined by fractionating the extracts into ribosomes and postribosomal supernatant (S150). An extract prepared without hemin or the S150 obtained from this extract prepared without hemin or the S150 obtained from this extract inhibits protein synthesis of the extract containing hemin by about 30%. The ribosomes prepared from extracts containing hemin are active in initiation of protein synthesis, whereas the ribosomes obtained from the extracts prepared without hemin show little or no initiation. These results have suggested that addition of hemin prevents the formation of an inhibitor of initiation in the S150 and at the same time protects from inactivation an initiation factor associated with ribosomes or ribosomal subunits. Addition of 2 mM GTP to HeLa extracts stabilizes the initiation activity, though to a smaller degree than hemin. The effects of hemin and GTP are not additive, suggesting that they may act on the same target molecule, though possibly by different mechanisms. The mechanism of action of GTP is discussed in view of similar observations made in the rabbit reticulocyte cell free system.


Studies on native ribosomal subunits from rat liver. Evidence for activities associated with native 40S subunits that affect the interaction with acetylphenylalanyl-tRNA, methionyl-tRNAf, and 60S subunits. The binding of the initiator tRNA Met-tRNAf, and of acetylphenylalanyl-tRNA, has been examined with rat liver 40S subunits derived from 80S ribosomes by dissociation with native 40S subunits sedimented from the postmicrosomal fraction and with native 40S subunits extracted with high salt-containing solutions. Binding of Met-tRNAf and acetylphenylalanyl-tRNA to derived and to salt-extracted native 40S subunits is observed in the presence of the appropriate polynucleotide template and a highly purified binding factor obtain from the soluble fraction of rat liver homogenates (R.L. IF-1). Native 40S subunits bind acetylphenylalanyl-tRNA in a reaction that requires poly(U) but not exogenous binding factor; however, Met-tRNAf is not bound to native subunits, even when supplemented with the soluble binding factor, or under conditions where factor-independent, high Mg2+-stimulated binding is observed with the derived and the salt-washed native 40S subunits. The extract obtained from native 40S subunits promotes the binding of acetylphenylalanyl-tRNA but not Met-tRNAf to derived and to salt-extracted native subunits. The addition of native 40S extract to incubations containing R.L. IF-1, Met-tRNAf, and derived 40S subunits, inhibits the formation of 40S-Met-tRNAf complex. These data suggest that the binding activity that is specific for 40S subunits and initiator tRNA, and an activity that inhibits the interaction with Met-tRNAf specifically, are both associated with native 40S subunits, and can be extracted from them by treatment with high salt-containing solutions. Derived 40S subunits react quantitatively with 60S particles to form 80S ribosomes which do not bind acetylphenylalanyl-tRNA with binding factor R.L. IF-1. Native 40S subunits react only partly with 60S subunits; about half of the native 40S subunit population forms 80S ribosomes which do not subsequently bind acetylphenylalanyl-tRNA; the remaining native 40S subunits which do not react with 60S particles bind acetylphenylalanyl-tRNA but to a lesser extent. When preformed native 40S-acetylphenylalanyl-tRNA complex is incubated with 60S subunits, about half of the subunits form an 80S-acetylphenylalanyl-tRNA complex, while the rest remains as 40S-acetylphenylalanyl-tRNA. The addition of native 40S subunit salt extract to incubations containing preformed 80S ribosomes dissociates the particles to subunits. These data suggest that in addition to the initiator tRNA binding activity and the activity that inhibits Met-tRNAf interaction, part of the native 40S subunit population also contains an activity that dissociates 80S ribosomes.


The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis). The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.


Isolation of soluble elastin from lathyritic chicks. Comparison to tropoelastin from copper deficient pigs. Tropoelastin was isolated from the aortas of chicks rendered lathyritic by treatment with beta-aminopropionitrile. The soluble elastin was judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and possessed an estimated molecular weight of 70000. Automated sequential analysis revealed that the N-terminal region of the chick tropoelastin is very homologous to tropoelastin isolated from copper-deficient piglets. N-terminal analysis of a trypsin digest of chick tropoelastin showed that tyrosine frequently is found adjacent to lysine residues. This positioning of tyrosine residues may be significant in terms of a possible regulatory role in elastin cross-link formation.


Equilibrium perturbation by isotope substitution. When malic enzyme is added to a mixture of malate-2-d, TPN, CO2, pyruvate, and TPNH at concentrations calculated to be at equilibrium, the TPNH level first drops and then increases slowly to its original level. This equilibrium perturbation is caused by slower cleavage of C-D than C-H bonds during hydride transfer as malate-2-d and TPNH are partly converted into TPND and malate-2-h in the process of establishing isotopic equilibrium. With malate-2-d, isotope effects for malic enzyme at pH 7.1 and malate dehydrogenase at pH 9.3 of 1.45 and 1.70-2.16 (depending on oxaloacetate level) were determined with this method, while the corresponding isotope effects on V/Kmalate and V for the chemical reactions were 1.5-1.8 and 1.0, and 1.9 and 1.5 for the two enzymes. The advantage of this method is its extreme sensitivity, and the lack of interference from various artifacts. The sensitivity is sufficient to permit determination of 13C and 15N isotope effects in favorable cases, and values of 1.031 for malic enzyme with 13CO2, and 1.047 for glutamate dehydrogenase with 15NH4+ have been determined. In the course of this work it was discovered that the equilibrium constants for oxidation by DPN, and oxidative decarboxylation by TPN are lower for malate-2-d than for malate-2-h by a factor of 0.76-0.82. Changes in Keq upon deuterium substitution, which are predicted by the calculations of Hartshorn and Shiner (1972), should be observed for many other reactions as well.


Dynamics of ligand binding to myoglobin. Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.


The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits: a solvent isotope effect. In the presence of specific monovalent cations (K+, Cs+, NH4+), inactive monomers of formyltetrahydrofolate synthetase associate to a catalytically active tetramer. The rate and extent of association of enzyme monomers prepared from C. cylindrosporum are enhanced 3.3-and about 50-fold, respectively, by the substitution of D2O for H2O. Both rate and equilibrium solvent isotope effects are due to a decrease in D2O of the dissociation constant of the monomer-cation complex. Analysis of rate and equilibria data obtained in solvent mixtures of varying deuterium/protium ratios indicates that the isotope effect may be due to the change in bonding of a single monomer proton during the association process. The data are most consistent with a model in which this proton is in a very weak potential in the cation-free monomer and is converted to a "normal" water-like proton in the monomer-cation complex.


Action of pancreatic phospholipase A2 on phosphatidylcholine bilayers in different physical states. 1. Saturated and unsaturated phosphatidylcholines, dispersed as liposomes in water, can be hydrolysed by phospholipase A2 from pig pancreas. A pure saturated phosphatidylcholine is hydrolysed only near its transition temperature. An unsaturated phosphatidylcholine is hydrolysed preferentially near its transition temperature, but hydrolysis can occur also above the transition temperature, albeit at a much lower rate. 2. An equimolar mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, which shows cocrystallization of the paraffin chains, is hydrolyzed between 25 and 40 degrees C with a maximum at 32 degrees C, in agreement with the calorimetric scan of the phase transition. 3. An equimolar mixture of dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine, which shows a monotectic behaviour, is hydrolysed at all temperatures. Hydrolysis is maximal at 0 and 40 degrees C, at which temperatures dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine undergo their phase transition, respectively. 4. Both in the mixture showing cocrystallization and in the mixture in which phase separation occurs, the phosphatidylcholine species with the shorter fatty acid chains is hydrolysed at a higher rate than the longer chain fatty acid species. 5. Hydrolysis is inhibited by the presence of cholesterol in liposomes prepared of saturated phosphatidylcholine. Inhibition is complete at a cholesterol concentration of 35 mol %. Subsequent addition of filipin and amphotericin B to the mixed cholesterol-phosphatidylcholine liposomes overcomes the inhibitory effect of cholesterol.


Photo-voltages of bilayer lipid membranes in the presence of cyanine dyes. The transmembrane photo-voltage waveforms induced by 10 different cyanine dyes absorbed to one side of bilayer lipid membranes are described. The membranes were prepared from lecithin, oxidized cholesterol, and mixed lecithin and oxidized cholesterol. An 8-mus flash illumination was used. Three dyes induced a photo-voltage which developed in a few milliseconds, then discharged in less than the membranes' resistance-capacitance time. Five dyes induced a photo-voltage which increased for much longer than the membranes' resistance-capacitance time. Two dyes did not induce any photo-electric effects. Models are presented which correlate the dye structure with the type of photo-voltage waveform induced.


The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes. This paper describes experiments showing the importance of the fatty acid chain length on the barrier properties of liposomal bilayers, prepared from saturated lecithins, under conditions of lateral phase separation. 1. Above the gel to liquid crystalline phase transition temperature, liposomes prepared from saturated lecithins with 14 or more carbon atoms per acyl chain exist as stable bilayers, which are practically impermeable to ions. 2. At temperatures well above the transition temperature dilauroyl phosphatidylcholine liposomes exhibited osmotic shrinkage, which was dependent on the ionic size of the solute used to bring about the osmotic gradient, indicating that the permeation through these less stable bilayers takes place mainly via individual diffusion of the permeating ions. 3. An enhanced release of trapped potassium from liposomes was demonstrated in the vicinity of the transition temperature. The extent of the increase, however, depended strongly on the length of the paraffin chain. 4. From measurements of the shrinkage behaviour of liposomes in the vicinity of the transition temperature it is concluded that the increased permeability decreases with increasing diameter of the permeating ion. This finding implies that the increased permeability at the transition temperature cannot be ascribed to "macroscopic" rupture of the liposomal membrane. The maximum permeability in the vicinity of the Tc is discussed in terms of probability and size distribution of statistical pore formation at the boundaries of liquid and solid domains.


Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus. The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.


Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus. The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [14C]mannose from GDP-[14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [14C]mannosyl-1-phosphorylundecaprenol. In contrast, this is the major mannolipid synthesized from GDP-[14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [14C]mannose from purified [14C]mannosyl-1-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.


Characterization of the passive and active transport mechanisms for bile acid uptake into rat isolated intestinal epithelial cells. The unstirred water layer has been shown to lead to an underestimation of apparent Km (Km(app)) values for active transport processes in intestinal whole tissue preparations. Isolated cells offer several potential advantages in the study of transport processes including a decreased diffusion layer of water adjacent to their absorptive membranes. Initial studies in cells isolated from rat intestine involving measurements of CO2 and lactate production and O2 consumption showed that overall metabolic pathways were functioning. Next, unidirectional uptake rates of bile acids across the isolated cell membrane were determined following correction for extracellular fluid contamination with a non-absorbable marker. Using epithelial cells isolated from jejunum P(app) for eight bile acid monomers varied from 24.9 (taurocholate) to 1563 (deoxycholate) nmol/min/100 mg protein/mM. From these data the incremental free energy changes for the addition of a hydroxyl, glycine and taurine group to the bile acid molecule were calculated to be 982, 1040 and 1464 cal/mol, respectively, values similar to those obtained after correction for unstirred water layer resistance in whole tissue preparations. Following subtraction of the passive component in isolated ileal cells complete kinetic curves for taurocholate and taurodeoxycholate yielded V(app) values of 109 and 70 nmol/min per 100 mg, respectively. Km(app) values of 0.24 mM (taurocholate) and 0.10 mM (taurodeoxycholate) are lower than usually recorded in whole tissue. Bile acid uptake into cells from ileum, but not jejunum, was affected by temperature, metabolic and competitive inhibition. These studies indicate that isolated epithelial cells are a metabolically viable, relatively purified intestinal preparation which discriminates between active and passive transport processes for bile acids under conditions where unstirred water layer artifacts are minimized.


Control of choline oxidation by rat-liver mitochondria. The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under "State-3" or uncoupled conditions, respectively. Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.


Photo-inactivation of system II centers by carbonyl cyanide m-chlorophenylhydrazone in Chlorella pyrenoidosa. In the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (4-10(-6) M), the S2 and S3 dark decays are accelerated and become biphasic with a first half-time of 0.6 s. The first fast phase of the decays does not correspond to a simple reduction of S2, S3 back to S0, S1 (i.e. to an acceleration of the deactivation reaction), but to a decrease in the number of oxygen-evolving System II centers. This photo-inactivation produced by CCCP is rapidly reversible in the dark.


Probing the kinetics of photosystem I and photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer. Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.


Reactions between primary and secondary acceptors of photosystem II in Chlorella pyrenoidosa under anaerobic conditions as studied by chlorophyll fluorescence. The kinetics of the fluorescence yield phi of chlorophyll a in Chlorella pyrenoidosa were studied under anaerobic conditions in the time range from 50 mus to several minutes after short (t 1/2 = 30 ns or 5 mus) saturating flashes. The fluorescence yield "in the dark" increased from phi = 1 at the beginning to phi approximately 5 in about 3 h when single flashes separated by dark intervals of about 3 min were given. After one saturating flash, phi increased to a maximum value (4-5) at 50 mus, then phi decreased to about 3 with a half time of about 10 ms and to the initial value with a half time of about 2 s. When two flashes separated by 0.2 s were given, the first phase of the decrease after the second flash occurred within 2 ms. After one flash given at high initial fluorescence yield, the 10-ms decay was followed by a 10 s increase to the initial value. After the two flashes 0.2 s apart, the rapid decay was not followed by a slow increase. These and other experiments provided additional evidence for and extend an earlier hypothesis concerning the acceptor complex of Photosystem II (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250-256; Velthuys, B. R. and Amesz. J. (1974) Biochim. Biophys. Acta 333, 85-94): reaction center 2 contains an acceptor complex QR consisting of an electron-transferring primary acceptor molecule Q, and a secondary electron acceptor R, which can accept two electrons in succession, but transfers two electrons simultaneously to a molecule of the tertiary acceptor pool, containing plastoquinone (A). Furthermore, the kinetics indicate that 2 reactions centers of System I, excited by a short flash, cooperate directly or indirectly in oxidizing a plastohydroquinone molecule (A2-). If initially all components between photoreaction 1 and 2 are in the reduced state the following sequence of reactions occurs after a flash has oxidised A2- via System I: Q-R2- + A leads to Q-R + A2- leads to QR- + A2-. During anaerobiosis two slow reactions manifest themselves: the reduction of R (and A) within 1 s, presumably by an endogenous electron donor D1, and the reduction of Q in about 10 s when R is in the state R- and A in the state A2-. An endogenous electron donor, D2, and Q- complete in reducing the photooxidized donor complex of System II in reactions with half times of the order of 1 s.


On the inhibitory action of cadmium on the donor side of photosystem II in isolated chloroplasts. The inhibitory effect of the Cd2+ in the electron transport of the isolated chloroplasts has been observed by measuring the oxygen uptake from the solution and the fluorescence induction. Cd2+ is found to be an inhibitor on the donor side of Photosystem II and its action site, as determined by experiments using hydroxylamine and exogenous Mn, is supposed to be on the water-splitting enzyme itself. Moreover, physicochemical and physiological studies indicate that only the ionic form of Cd is acting at the level of the manganoprotein. It is not possible, from this work, to define precisely in which form Cd is taken up through the thylakoid membranes.


Inhibition of oxygen evolution following illumination of Chlorella cells with far-red light. The effect of brief far-red illumination on Chlorella pyrenoidosa cells has been investigated. An inhibition of oxygen evolution occurs 20 to 30 s after the end of the far-red illumination. This inhibition occurs in a step following the initial charge separation process by the System II centers. It is reversible in the light through a purely photochemical process.


Intermembrane electron transport in the absence of added water-soluble carriers. Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b5 reductase or cytochrome b5 has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems "intact mitochondria - mersalyzed microsomes" and "mersalyzed mtiochondria - untreated microsomes". No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b5 reductase and/or cytochrome b5, localized in microsomal and outer mitochondrial membranes.


New chlorophyll-b forms in a chlorophyll-detergent photosynthetic model system. The absorption spectra of chlorophyll b in Triton X-100 micelles at room temperature are superpositions of components with absorption maxima at 640.8, 648.9, 659.5, 669.6, 682.1 and 695.7 nm, obtained from Gaussian analysis of the spectra. The last four forms strongly overlap the chlorophyll a forms of this system obtained with maxima at 659.3, 667.6, 674.3, 680.8, 686.5, 692.8, 701.9, 713.6 and 722.0 nm. Since the in vivo chlorophyll a forms practically coincide with the forms found in this system, the possible existence of in vivo overlapping chlorophyll b and a forms eventually should be taken into consideration. In this case, however, the Gaussian analysis of in vivo absorption bands in itself in the proper spectrum range cannot discriminate between chlorophyll a and b components.


Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the water-splitting activity in photosynthesis. Plant materials (intact leaves, chloroplasts or subchloroplast particles) pre-illuminated at a low temperature (e.g. -60 degrees C) were rapidly cooled to -196 degrees C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as ZV, A, B1, B2 and C bands. The A, B1, B2 and C bands appeared at constant temperatures, -10, +25, +40 and +55 degrees C, respectively, being independent of the illumination temperature, but the ZV band appeared at a variable temperature slightly higher than the illumination temperature. The B1 and B2 bands were absent in the thermoluminescence profiles of samples devoid of the oxygen-evolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.


Photophosphorylation and related properties of reaggregated vesicles from spinach photosystem I particles. The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH+4 are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.


Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide. In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO2 reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of 18O2 during reduction of CO2 was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO2 assimilation resulted in the formation of H2O2. This was indicated by a large stimulation of CO2 reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H2O2 was shown to be at the level of phosphoglycerate formation. Evidence for electron transport of oxygen was also obtained from 14CO2 experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO2 reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO2 and oxygen during photosynthesis.


Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems. The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.


Effect of phosphatidylcholine on fatty acid and cholesterol absorption from mixed micellar solutions. Using the experimental model of the everted sac prepared from rat jejuna, kinetic studies on [14C]oleic acid uptake from bile salt micelles were conducted in the presence and absence of phosphatidylcholine. The concentration of oleic acid was varied between 0.625 and 5 mM. At every level of fatty acid concentration studied the addition of 2 mM phosphatidylcholine produced a significant inhibition of fatty acid uptake. It was further noted that the intact phospholipid molecule was required for this effect as lysophosphatidylcholine produced little, if any, inhibition of [14C]oleic acid uptake. The effect of varying the concentration of phosphatidylcholine on fatty acid uptake was also studied. The degree of inhibition was noted to be correlated grossly with media concentrations of this phospholipid although the decrease of fatty acid uptake was not strictly proportional to concentration of this material in the medium. Studies were also performed analyzing in vitro absorption of [14C]oleic acid and [3H]cholesterol simultaneously from mixed micelles composed of sodium taurocholate, oleic acid, monoolein and cholesterol. Control medium contained no phospholipid while experimental medium contained either diester or diether phosphatidylcholine, 2 mM. Both types of phosphatidylcholine caused significant inhibition of fatty acid and cholesterol uptake. In vivo absorption studies were also performed using the isolated jejunal segment technique. A mixed micellar solution containing [3H]cholesterol and [14C]oleic acid was used as the test dose. Phospholipid in the test dose for controls was supplied as lysophosphatidylcholine and for experimentals it was in the form of diether phosphatidylcholine. Significantly less radioactively labeled cholesterol and fatty acid was absorbed by experimentals as compared to controls over a 10-min period. It is concluded that the intact molecule of phosphatidylcholine inhibits intestinal uptake of cholesterol and fatty acid from mixed micellar solutions under both in vitro and in vivo conditions.


Incorporation and subcellular distribution of an unnatural phospholipid base-analog, N-isopropyl-ethanolamine, in rat liver. An unnatural phospholipid, phosphatidyl-N-isopropylethanolamine, was isolated from rat liver after intraperitoneal injections of N-isopropylethanol-amine; it was identified on the basis of enzymic, chemical, and chromatographic analyses. Although this phospholipid was formed at the expense of phosphatidylcholine and phosphatidylethanolamine, its fatty acid composition did not resemble either of these lipids. Microsomes, mitochondria, and plasma membranes contained significant amounts (up to 9%) of this unusual phospholipid. Radioisotope incorporation experiments suggest that the N-isopropylethanol-amine containing phospholipid is rapidly equilibrated between microsomes and mitochondria and more slowly with surface membranes.


Regulation of hepatic fatty acid synthetase in the obese-hyperglycemic mutant mouse. Regulation of fatty acid synthetase has been studied in the obese-hyperglycemic mouse and compared with regulation in non obese, littermate control animals. The mechanisms underlying the regulatory changes were defined by immunochemical techniques. Several major conclusions are justified from the data obtained: (1) Although the hepatic specific activity of fatty acid synthetase is higher in obese than in non obese animals pair-fed chow, no difference in hepatic activities is apparent in animals pair-fed the fat-free diet; (2) The higher enzymatic activity in obese animals fed chow is related to a higher content of enzyme, and this higher content is associated with a higher rate of enzyme synthesis; (3) The decrease in hepatic synthetase activity with starvation is distinctly more striking in non obese than in obese animals, and the changes in activity reflect changes in content of enzyme; (4) With starvation there is a decrease in synthesis of enzyme in obese and non obese animals, but only in non obese animals is there also a marked increase in the rate of synthetase degradation (t1/2 = 24 h during starvation, t1/2 = 76 h during normalfeeding); (5) Refeeding starved mice a fat-free diet results in a more striking increase in hepatic synthetase activity in non obese than in obese animals; (6) Administration of triiodothyronine causes a more marked increase in hepatic synthetase activity in non obese than in obese animals. The data thus define a variety of differences in regulation of hepatic fatty acid synthetase in mutant and normal animals. The roles of enzyme synthesis and degradation in the etiology of these differences are defined, and possible mechanisms underlying regulation of synthetase synthesis and degradation in normal mammalian liver are suggested by the observations.


A novel sulfonolipid in diatoms.

A novel sulfonolipid in diatoms. A new sulfonolipid has been isolated from a non-photosynthetic diatom, Nitzschia alba, by thin-layer and column chromatography on silicic acid, and characterized by 35S-labeling, mobility on thin-layer chromatography, infrared and NMR spectroscopy and products of hydrolysis, as a ceramide sulfonic acid (N-acyl sphingosine-1-sulfonic acid). The long-chain base moiety was shown by identification of the products of periodate or periodate-permanganate oxidation to consist of a C18-trans-sphingosine backbone linked directly by a C-S linkage through C1 to a SO3 group. The N-acyl groups were mainly isoheptadecanoic (64%) and palmitic (26%) acids.


Purification and properties of xanthine dehydrogenase from Pseudomonas acidovorans. Xanthine dehydrogenase (EC 1.2.1.37) from Pseudomonas acidovorans has been purified to near homogeneity (approx. 65-fold). The enzyme has a molecular weight of about 275 000. Electrophoresis in gels containing sodium dodecyl sulphate showed the presence of two types of subunit with molecular weights of about 81 000 and 63 000. Thus the intact molecule probably contains two of each type of subunit. Xanthine and hypoxanthine are good substrates, and NAD+ is an effective electron acceptor. With xanthine and NAD+ as substrates the purified enzyme has a specific activity of about 20 mumol NADH formed/min per mg protein. Michaelis constants for xanthine and NAD+ are 0.07 and 0.12 mM, respectively, and for hypoxanthine and NAD+ 0.29 and 0.16 mM, respectively.


Separation of active enzyme components from the fatty acid synthetase of chicken liver. Fatty acid synthetase (EC 6.2.1.-) of chicken liver was dissociated into half-size subcomplexes and then separated into three protein fractions by the preparative disc-gel electrophoresis technique. The anodal protein (Fa) of a molecular weight of approx. 6000 contains the prosthetic group, 4'-phosphopantetheine. It binds acetyl group from acetyl-CoA and is identified as the acyl carrier protein component. The slower moving proteins (FI and FII) correspond to the subcomplexes resolved by the analytical method (Y.n, S.L. and Hsu, R.Y. (1972) J. Biol. Chem 247, 2689--2698). Both contain acetyl transacylase and palmityl-CoA deacylase activities, but only FI contains beta-ketoacyl reductase activity. Active acetyl transacylase and palmityl-CoA deacylase components were obrained by the sucrose density centrifugation technique in a broad 3 S protein band from the FI fraction, following dissociation at 4 degrees C for 12 days. Slight modification of the electrophoresis conditions yields a homogeneous 1.55 S beta-ketoacyl reductase component.


Binding of homogeneous cytochrome b5 to rat liver microsomes. Effect on N-demethylation reactions. Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.


Effect of aggregation on the kinetic properties of aspartate aminotransferase. In this investigation the steady-state kinetic parameters of the alpha subform of aspartate aminotransferase (EC 2.6.1.1) were determined in 0.2 M Tris - HCl, pH 8.0, at 25 degrees C. The kinetic parameters for both the forward and reverse reactions were determined under conditions where the enzyme is monomeric, while only the steady-state parameters associated with the forward reaction could be determined under conditions where the enzyme is dimeric enzyme decreased relative to that of monomeric enzyme, 245 versus 360 s(-1) while the Km for aspartate increased, 3.3 versus 2.6 mM. No significant change in the Michaelis constant for ketoglutarate was observed. The steady-state parameters of dimeric enzyme are slightly altered in 0.1 M Na4 P2O7, pH 8.0, the catalytic center activity and Michaelis constant for ketoglutarate being slightly larger. From the dependence of the initial velocity on enzyme concentration the dissociation constant for the monomer-dimer equilibrium is estimated to be 2 - 10(-8) M. A similar value of the dissociation constant was estimated from Sephadex gel filtration experiments.


Peptide hydrogen exchange rates in Streptomyces subtilisin inhibitor. The exchange reaction of the peptide NH protons of a microbial protease inhibitor (Streptomyces subtilisin inhibitor) with deuterium atoms in 2H2O (p2H 6.8) has been studied by proton magnetic resonance in the temperature range 56-71 degrees C. Both slowly and rapidly exchanging processes have been observed. The number of slowly exchanging protons is estimated to be 25 +/- 2 per subunit of the protein molecule. The decay of the slowly exchanging proton signals follows a single time-exponential function at each temperature. The observed first-order rate constants have been analyzed to give the denaturated fraction of the protein as a function of temperature with a consequent enthalpy (56 kcal/mol) and an entropy (137 cal/degree per mol) of denaturation. The results indicate the high conformational stability of this protein against heat denaturation.


Mechanism of action of heparin through thrombin on blood coagulation. It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational change in the enzyme, facilitating a complex formation between thrombin and antithrombin (Machovich, T., Blaskó, Gy. and Pálos, L. (1975) Biochim. Biophys. Acta 379, 193-200). This hypothesis seems to have been proved. Modification of arginine residues in thrombin did not result in decreased thrombin activity and decreased sensitivity to antithrombin, whereas the heparin sensitivity of the enzyme and the thrombin-antithrombin reaction were diminished.


Hemoglobin-Strumica or alpha 2 112(G19) His replaced by Arg beta 2. (With an addendum: hemoglobin-J-Paris-I, alpha 2 12(A10) Ala replaced by Asp beta 2, in the same population). Hemoglobin-Strumica has been observed in five members of a Macedonian family. Histidyl residue in position 112 of the alpha chain of this variant is replaced by an arginyl residue. Two other variants (hemoglobin-Dakar and hemoglobin-Hopkins-2) in which this histidyl residue has been replaced by a glutaminyl and by an aspartyl residue, respectively, have been described (Rosa et al. (1968) 12 thCongr. Int. Soc. Haematol. New York, abstract, p. 73 and Charache, S. and Osterag, W. (1970) Bloodt 36, 852). The hemoglobin-Strumica heterozygotes have minimal hematological changes although this may not necessarily be associated with the hemoglobinopathy. Subjects heterozygous for hemoglobin-Dakar have a mild hemolytic anemia and hemoglobin-Hopkins-2 heterozygotes exhibit minimal hematological changes.


Hydrophobic interaction chromatography on non-charged Sepharose derivatives. Binding of a model protein, related to ionic strength, hydrophobicity of the substituent, and degree of substitution (determined by NMR). A series of agarose gels, substituted with hydrophobic  groups, has been synthesized and used for binding studies with the coloured model protein, phycoerythrin. The degrees of substitution for the derivatives can easily be estimated with proton magnetic resonance (NMR). It has been found that the capacity of the derivatives for phycoerythrin increases with increasing hydrophobicity of the substituent, degree of substitution and increasing ionic strength. For column experiments the degree of substitution should lie in the range 40-100 mmol substituent/mol galactose. When it is excessively high, the flow characteristics of the columns are unsatisfactory and difficulties to achieve complete desorption may arise.


Human pituitary lutropin. Isolation, properties, and the complete amino acid sequence of the beta-subunit. The separation of alpha and beta subunits from human pituitary lutropin is described, and the complete amino acid sequence of the beta subunit is proposed. It consists of 115 amino acids with serine and glycine at the amino and carboxyl termini, respectively. The single carbohydrate moiety is linked to asparagine in position 30 and the single tryptophan of the lutropin molecule is located at position 8. The two methionine residues in lutropin-beta are in positions 41 and 42. In addition to COOH-terminal heterogeneity, evidence for internal peptide cleavages was observed.


The isolation and characterization of alpha-keratin microfibrils. A method of isolating alpha-keratin microfibrils which avoids the degradation previously associated with the use of chemical, physical or enzymic procedures has been developed. Electron microscope studies of the isolation procedure establish that the microfibrils originate from the presumptive cortical cells. A purification procedure, monitored by electron microscopy, has enabled microfibrils to be isolated on a scale sufficient for chemical characterization. The amino acid composition of the microfibrils is very similar to that of low-sulphur protein fractions extracted from a range of hard mammalian keratins and thus provides direct experimental evidence for the assumption that the low-sulphur proteins comprise the microfibril in alpha-keratin.


Isolation and characterization of several glycoproteins from human colostrum whey. Five homogeneous closely related glycoproteins were isolated from human colostrum whey, and their molecular weights and carbohydrate contents ranged from 26 000 to 35 000 and from 50 to 80%, respectively. All had the Lactobacillus bifidus var. Pennsylvanicus growth-promoting activity, which was not substantially altered by desialization. Neither human colostrum, milk or the glycoproteins had any cellular immunity-mediating activity. Degradation of the carbohydrate moieties of these glycoproteins indicated that each contained non-identical carbohydrate chains.


Inhibition of human thrombin assessed with different substrates and inhibitors. Characterization of fibrinopeptide binding interaction. The activity of human thrombin has been assessed with fibrinogen, N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, N-alpha-benzoyl-arginine-p-nitroanilide, N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and p-nitrophenylacetate: increased rates of hydrolysis were found for N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide compared to N-alpha-benzoyl-arginine-p-nitroanilide and p-nitrophenylacetate. Phenylmethyl sulfonyl fluoride and N-alpha-tosyl-L-lysine chloromethyl ketone inhibited, to the same degree, the activity toward each substrate. Inclusion of N-alpha-tosyl-arginine methyl ester in the phenylmethyl sulfonyl fluoride reaction mixtures protected the enzyme from inhibition as shown with N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. N-Acetylimidazole inhibited the activity towards fibrinogen, N-alphrosine-p-nitrophenyl ester to varying degrees. Inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide was completely reversible with neutral hydroxylamine, whereas coagulant activity towards fibrinogen was only partially regained. Human fibrinopeptide A inhibited activity toward N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. The mode of inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide by fibrinopeptide A was non=competitive (K1 = 3.02.10(-5) M), whereas N-alpha-toysyl-arginine methyl ester was a competitive inhibitor of this substrate (K1 = 2.6.10(-5) M). These studies demonstrate more than one binding domain for fibrinogen on human thrombin.


Effect of modification on physicochemical and biological properties of haptoglobin. VI. Reaction with azlactone of p-nitrobenzoyl-valine. The azlactone of p-nitrobenzoyl-valine (Nbz-Val) has been used for modification of xi-amino groups of lysine in haptoglobin type 1-1, in hemoglobin, and in the haptoglobin-hemoglobin complex. By the use of this reagent 95% of amino groups in haptoglobin and 90% in hemoglobin have been blocked without any changes in peroxidase activity of the formed complexes: Nbz-Val.haptoglobin with hemoglobin, Nbz-Val. hemoglobin with haptoglobin, and Nbz-Val.(haptoglonin-hemoglobin). After reduction and reoxidation, Nbz-Val.haptoglobin was found to retain 90% of peroxidase activity when complexed with hemoglobin. Beta chains separated either from haptoglobin or Nbz-Val.haptoglobin showed 15% of peroxidase activity in the complex with hemoglobin, alpha chains of the same origin were completely inactive. Whereas recombination of haptoglobin from alpha and beta chains resulted in 42% hemoglobin-binding capacity, renaturation of Nbz-Val.haptoglobin from separated subunits was found to proceed with almost 100% yield. In immunodiffusion with rabbit anti-haptoglobin or anti-Nbz-Val.haptoglobin sera, preparations of haptoglobin and Nbz-Val.haptoglobin after reduction and reoxidation or after recombination from separated subunits gave similar precipitation arcs showing the reaction of immunological identity.