Myocardial infarction in car assembly workers. The incidence of myocardial infarction and the return to work for survivors were studied among the employees of an English car assembly plant by analysing 12 811 medical records of persons employed during the seven years between January 1966 and December 1972. The standardized morbidity ratio of myocardial infarction found in this study calculated on the basis of incidence rates reported by Kinlen )1973) for the Oxford community in which the factory was situated was 90. The standardized morbidity ratio from production line workers only was 66 and that for the monthly paid staff 272. Of the production line workers who survived the attack 22 (90%) returned to their previous jobs without undue difficulty and with two exceptions within four months of the onset of their illness; there was no relation between length of absence and age at the time of attack. These findings suggest that workers in mass production jobs such as car assembly are not special risk from myocardial infarction, and most of those who survive a heart attack are able to return to their former work. Taken with Kinlen's (1973) study and that of Armstrong et al. (1972) in Edinburgh, they also bear out mortality data by indicating that in Oxfordshire the incidence of coronary heart disease is lower than the British average.


Significance of irregular small opacities in radiographs of coalminers in the USA. The purpose of this study was to determine the factors that are associated with the occurrence of small irregular opacities in the chest radiographs of coalminers, and whether the lung function of miners with irregular opacities differed from that of miners with small rounded opacities, a mixture of small rounded and irregular opacities, or an absence of opacities. A subsample of 6166 coalminers was selected from 9076 miners who had been examined by the US Public Health Service as part of the National Study of Coalworkers' Pneumoconiosis. The subsample consisted of 4479 smokers and 1687 non-smokers. The chest radiograph of each miner was classified according to the UICC/Cincinnati Classification.


Follow-up of ventilatory lung function in a group of cement workers. In a group of 160 active cement workers and 80 control workers selected on the basis of having or not having symptoms of chronic bronchitis, forced vital capacity (FVC) and one second expiratory volume (FEV 1-0), both corrected for age and height, and ratio of one second forced expiratory volume to forced vital capacity (FEV 1-0/FVC (%)) were measured on two occasions with an interval of four and eight years respectively.


Induction of fibrogenesis by lung antibody-treated macrophages. Using a modification of an in vitro model of fibrogenesis, lung connective tissue antibodies have been shown to stimulate macrophages to release a collagen stimulating factor acting on fibroblast target cells. This stimulation was measured by increased hydroxyproline production from fibroblasts that had reached stationary growth phase. In subcytotoxic amounts, this antibody had no such effect directly on fibroblasts. These findings further illustrate the value of studying fibrogenesis by the in vitro method.


Biochemical changes during the initial stages of industrial lead exposure. Biochemical measurements were carried out on 20 workers entering employment in a lead industry. Blood lead (Pb-B) urinary delta-aminolevulinic acid (ALA), and coproporphyrin (CP) levels were determined before entry and then at approximately weekly intervals for the first 12 weeks of employment. Levels of blood lead began to increase in all men within one week rising from an average pre-employment figure of about 1-1 mumol/l (22 mug/100 ml) to a value of about 2-9 mumol/l (60 mug/100 ml) at three weeks. Subsequently there was only a small increase in Pb-B values levelled for the rest of the period.


Activity of erythrocyte delta-aminolevulinic acid dehydrase and its change by heat treatment as indices of lead exposure. The effect of heat treatment on erythrocyte delta-aminolevulinic acid dehydrase (ALAD) activity in the blood of human subjects was examined in relation to their lead exposure.


Plasma concentrations of the oxime Pralidoxime Mesylate (P2S) after repeated oral and intramuscular administration. The use of the oxime P2S as intravenous therapy for organophosphorus anticholinesterase poisoning is well known. In emergency situations this route of administration may prove impractical due to severe symptoms of anticholinesterase poisoning and therefore the intramuscular route is to be preferred. The absolute intramuscular dose of P2S per man, recommended as necessary for adequate therapy of anticholinesterase poisoning, is 500 mg. In practical situations this dose may have to be repeated at intervals resulting in overdosage, and therefore, the clinical side-effects which this regimen might have on normal subjects has been determined. It has also been suggested that where organophosphorus anticholinesterase compounds are handled continuously for many months  in the year, for example, in crop spraying and in industry, P2S might be taken prophylactically.


Absence attributed to sickness in oil tanker crews. Absences attributed to sickness were investigated in 1410 deck and engine-room crew members during a period of two years and five months. The mean frequency of absences was 0-23 per man year, with a mean duration per absence of 41 days. The absence frequency varied with both rank and place of work. Altogether 23% of deck officers serving throughout the study and 43% of engine-room ratings had one or more absences. Spells of absence in young officers were five times more frequent when they were on leave than at sea. In the younger officers more than half of all spells that were initiated while on leave occurred at the end of the leave period. The contrasting environments of ship and shore allow the relative importance of effects on absence frequency of the work and home environment and of medical and social factors to be considered separately.


The sleep habits, personality and academic performance of medical students. The academic performance of 104 fourth-year medical students was assessed in relation to their sleep habits reported in a questionary and their scores on the Minnesota Multiphasic Personality Inventory. Poorer academic performance was related significantly to later times of waking up in the morning, particularly at weekends, and to subjectively poorer quality sleep, but not to the amount of sleep usually obtained. Poor academic performance was related also to scores on scales 3 (hysteria), 4 (psychopathic deviate) and 8 (schizophrenia) of the MMPI. Simple enquiries about sleep habits may make it easier for students who are at greatest risk of academic failure to be identified and helped.


The prediction of medical student performance using in-course assessment. The value of in-course assessment at King's College Hospital Medical School at predicting student performance at final qualifying examinations in medicine and surgery has been analysed. Only a small proportion of the variation in marks at finals could be explained by the factors studied. Both the MCQ and firm report methods of internal assessment used in the first clinical year were equally valuable at predicting final outcome. Performance at 2nd MB BS, the possession of another degree and the sequence of firm attachment in the first year have little predictive value. Female students are predicted to perform better in medicine than in surgery. The development of a more standardized quantifiable firm assessment looks potentially more valuable at predicting performance than refining the first year MCQ examinations.


The attainment of specified objectives by medical students in different learning environments. Medical students were provided with behavioural objectives for the problem solving phase of their curriculum. They were assigned for 11 weeks to various hospital settings that differed in the presence or absence of structured classes, the expertise of the teaching staff, the amount of instruction time and the types of learning experiences. At the end of that time, the students took the same examination to measure attainment of the objectives. The only variable that was associated with significantly different examination scores was presence or absence of structured classes.


The use of computerized patient management problems in a certifying examination. The 1974 paediatric certification process of the Royal College of Physicians and Surgeons of Canada included four CPMPs, in addition to the conventional MCQs and orals. The CPMP's were successfully administered to 160 candidates in eight cities across Canada on the same day. The examination was judged by the candidates to be a better test of clinical skill than MCQs. The CPMP examination did not penalize French-speaking candidates, was economically feasible, and a good security risk. Correlations between MCQ, orals and CPMPs, indicated that each examination measured some aspects of paediatric competence not tested by the others.


Teaching psychologists to teach psychology: the improvement of teaching skills in health service professions. The need to improve the quality of teaching received by health service professionals is apparent from several recent reports. A short course of approximately 10 class hours is described, as used to train clinical psychologists in teaching method, with particular reference to teaching nurses. Problems of lecture content and preparation are discussed, emphasis is laid on the use of varied and active teaching methods, and the need for feedback from the students. The course was well received by students, and acceptable into an already crowded curriculum.


Teaching the teachers.

Teaching the teachers. This and the following two papers describe historically the development of courses for general practice teachers in Manchester from 1966. The first paper describes the early attempts to fashion an appropriate course, discusses the content of such courses and the methods used. The second and third papers then discuss a course on non-directive behavioural counselling as an educational tool and describe the evolution of a cycle of four courses, each dealing with separate aspects of teaching. The emphasis throughout is on person to person teaching, it being considered that this is the commonest teaching/learning situation in general practice. The second part of the first paper goes on to discuss the logistic needs for the preparation and maintenance of teachers in general practice.


A teaching methods course in Manchester for general practitioner teachers. A teaching-methods course for general practitioner teachers is described. It lasts for 20 days, split into four 5-day residential sections held at intervals of 6 months. The sections are respectively entitled person-to-person teaching, small group teaching, teaching behaviour, and a micro-training laboratory. They recur in a cycle. The approach in each section is that of experimental learning--necessarily, since an aim of the course is to enable the members to apply the same approach in their own teaching. The methods of three of the sections are given in some detail. The person-to-person teaching section, concerned mainly with the learning of counselling skills, is dealt with fully by Long et al. (1976), and is here presented very briefly. The limited evaluation and assessment procedure, and their results, are offered together with certain general conclusions.


Assessment by dual examination of the teaching of respiratory diseases. This study was designed to test the effectiveness of the teaching programme in respiratory diseases. It showed that those students who did not have a clinical attachment in the speciality were at a disadvantage when compared with their fellows provided with a clinical attachment.


Cigarette smoking and fetal breathing movements. Cigarette smoking caused a reduction in the incidence of fetal breathing movements in normal and abnormal pregnancies. The size of the reduction varied, being greatest in small-for-dates pregnancies and pregnancies complicated by fetal distress in labour and least in pre-eclamptic pregnancies. The fall in the amount of fetal breathing movements was significantly related to the rise in maternal plasma nicotine after smoking but was unrelated to the rise in barboxyhaemoglobin. Smoking non-nicotine (herbal) cigarettes produced increases in carboxyhaemoglobin concentrations similar to those observed after smoking tobacco cigarettes, and was not associated with a fall in the incidence of fetal breathing movements. Chewing gum containing nicotine produced rises in plasma nicotine concentration similar to those observed after smoking tobacco cigarettes and was associated with a significant reduction in the incidence of fetal breathing movements. Hence nicotine appeared to be the factor in cigarette smoke responsible for the reduction in the incidence of fetal breathing movements. Nicotine was present in the cord blood of infants whose mothers smoked. The possible mechanism by which nicotine caused a reduction in the incidence of fetal breathing movements and its possible relevance to the detrimental effects of smoking on the fetus are considered.


Dolichocephaly--a source of error in serial cephalometry. Two patients are presented in whom ultrasonic cephalometry suggested intrauterine fetal growth retardation. The respective newborn infants were normally grown and had dolichocephaly with a normal head circumference. Dolichocephaly should be considered when cephalometry alone suggests a diagnosis of placental insufficiency.


The relation between ease of forceps delivery and speed of cervical dilatation. An attempt has been made to predict a difficult forceps delivery. The duration of the 7 to 10 cm cervical dilatation interval was measured from the completed cervimetric chart in 952 consecutive patients who delivered spontaneously between December 1973 and September 1974. The 7 to 10 cm cervical dilatation intervals of this group were compared with those of 378 consecutive patients in whom forceps were applied with the fetal head in the occipito-anterior position and 83 consecutive patients where Kielland's forceps rotation from the occipito-tranverse or occipito-posterior position was performed. The forceps deliveries were graded as 'easy', 'moderately difficult', or 'difficult'. In only 5% of the spontaneous delivery group did the 7 to 10 cm cervical dilatation interval exceed two hours. In the occipito-anterior and Kielland's forceps groups an 'easy' delivery could be expected if the 7 to 10 cm cervical dilatation interval was less than two hours. The greater this interval increased beyond two hours, the greater was the proportion of 'moderately difficult' and 'difficult' forceps deliveries.


Platelets and intrauterine growth retardation in pre-eclampsia. In pre-eclampsia, but not essential hypertension of pregnancy, reduced maternal levels of circulating platelets were found to correlate with intrauterine growth retardation. This suggests that disseminated intravascular coagulation and fibrin deposition contribute to the placental damage of pre-eclampsia.


Circulating levels of fibrin/fibrinogen degradation fragment E measured by radioimmunoassay in pre-eclampsia. Levels of fibrin/fibrinogen degradation products have been measured by a sensitive and specific radioimmunoassay in pregnancies associated with pre-eclampsia or essential hypertension. The incidence of elevated levels was low (2-4%) in mild and moderate cases, and rather higher (30%) in severe cases. In no patients were the levels consistently elevated prior to the onset of clinical signs.


Studies on the effect of maternal pre-eclamptic toxaemia on placental weight and on head size and birth weight of the newborn. The influence of pre-eclamptic toxaemia (PET) on head size and birth weight of newborn infants and placental weight was studied in single live births after 35 to 42 weeks gestation from mothers with or without toxaemia. There were no significant differences in the distribution of mean birth weight, head size, placental weight, head size/birth weight ratio or placental weight/birth weight ratio between the toxaemic and non-toxaemic groups. There were significant differences between toxaemic and non-toxaemic mothers as regards parity, height and smoking habits. Since such maternal characteristics influence birth weights a correction was applied for them. The influence of maternal mid-term weight and sex of the infants was also taken into account. No significant differences in corrected birth weights, between toxaemic and non-toxaemic groups were detected on further analysis. It is suggested that the present obstetric management has prevented the retarding effect of pre-eclamptic toxaemia on fetal and placental growth by reducing the severity and duration of the illness.


Plasminogen activators in the endometrium of women using intrauterine contraceptive devices. Plasminogen activators in the endometrium before and after the insertion of an intrauterine contraceptive device (IUCD) were studied; an increase in the concentration was found following IUCD insertion in 16 of the 20 women. This change will enhance endometrial fibrinolysis and may explain the increased menstrual bleeding which usually follows insertion of an IUCD.


Practolol peritonitis presenting as a pelvic mass. A case of sclerosing fibro-peritonitis occurring as a result of practolol therapy is presented. This may occur months after cessation of the practolol because of other side effects and may present to the gynaecologists as a pelvic mass.


Studies in the involvement of prostaglandins in uterine symptomatology and pathology. The concentrations of prostaglandin E2 and F2alpha were measured by radioimmunoassay in samples of endometrial tissue from 155 women. The results showed that the concentration of prostaglandins E2 (ng/100 mg of wet tissue; mean +/- SD) in tissue from apparently healthy women with regular menstrual cycles was 79-3 +/- 9-9 during the menstrual period. The level in the proliferative and early secretory phases fell to 11-8 +/- 4-0 and 13-3 +/- 8-3 respectively. During the late secretory phase there was a significant increase to 20-1 +/- 10-8. The corresponding values from patients with fibromyomata and without menorrhagia were similar. However, the concentration in tissue from patients with fibromyomata and menorrhagia was significantly higher (63-9 +/- 17-9) during the secretory phase of the uterine cycle. In addition, the amount was significantly higher in the groups with dysmenorrhoea (19-0 +/- 4-5 proliferative; 68-0 +/-18-15 secretory) and irregular uterine bleeding (78-3 +/- 59-0 proliferative; 87-4 +/- 64-9 secretory). The mean concentration was higher in cases of endometriosis, and significantly elevated in endometrial carcinoma. The results for prostaglandin F2alpha showed a similar pattern, but the changes were less marked in patients with irregular dysfunctional bleeding. The findings are discussed.


Factors associated with spontaneous pre-term birth. Factors associated with spontaneous pre-term birth in 283 singleton pregnancies were compared with those present in a total population of 16 994 women at risk studied in the First British Perinatal Mortality Survey. It was shown that the risk of spontaneous pre-term birth was related to low maternal age, low maternal weight, maternal smoking, low social class, illegitimacy, threatened abortion, and a previous history of antepartum haemorrhage, perinatal loss, or low birth weight livebirths.


Antenatal identification of women at high risk of spontaneous pre-term birth. A means of assessing the risk of a spontaneous pre-term birth in any individual woman presenting at the antenatal clinic is described. A composite score can be obtained by the multiplication of relative risks for various factors. The effectiveness of the method is discussed.


Coagulation and fibrinolysis in pregnancies complicated by fetal growth retardation. Saline extracts were made from portions of 17 normal placentae and from 8 placentae from pregnancies complicated by fetal growth retardation, but not hypertension. The ability of these extracts to inhibit urokinase-induced fibrinolysis was measured using a fibrin plate technique. Placental extracts from pregnancies complicated by fetal growth retardation exhibited greater inhibition of urokinase-induced fibrinolysis. There was no evidence of disseminated intravascular coagulation in these patients, but certain coagulation factors in the peripheral blood were raised.


Longitudinal study of the free cortisol index in pregnancy. Total plasma cortisol, the 'cortisol resin uptake ratio' (cortisol RUR), and 'free cortisol index' (FCI) were estimated in 23 pregnant women from 12 weeks to term and at six weeks post partum. Total plasma cortisol rose continually throughout pregnancy; the cortisol RUR rose up to 20 to 23 weeks and then fell slowly and the FCI also rose although more slowly than the total cortisol. All parameters fell to non-pregnant levels at six weeks post partum. It is confirmed that a true rise in metabolically active cortisol occurs during pregnancy.


Iron stores in pregnant patients with anaemia and haemoglobin S. Marrow aspiration in 22 pregnant women with haemoglobin S revealed a high incidence of iron deficiency. Eighteen patients showed depleted iron stores while depots were adequate in four patients. The practice of denying iron to pregnant women with haemoglobinopathies is not recommended; nor is the use of prophylactic iron in all patients with sickle cell disease or trait advised. It is suggested that it is better to diagnose and treat the iron deficiency specifically.


Tissue oxygenation and red cell 2,3-diphsophoglycerate in normal and abnormal pregnancy. Maternal tissue oxygenation was reflected by the level of red cell, 2,3-diphosphoglycerate (DPG) was measured before, during and after normal pregnancy. Following an initial fall at the beginning of pregnancy there was a significant rise in the mean level of DPG during pregnancy with an abrupt fall in the puerperium. The mean red cell DPG level in the third trimester of pregnancies complicated by pre-eclampsia and diabetes was not statistically different from the normal but the mean value of all pregnancies in which the fetus was stillborn or growth retarded was significantly lower (p less than 0-001). The possible mechanism of the changes in normal and abnormal pregnancy is discussed and it is suggested that the measurement of red cell DPG in the third trimester of pregnancy may prove to be a useful parameter of placental oxygenation.


Individual free fatty acids in amniotic fluid and in plasma of pregnant women. Individual free fatty acids (FFA) were measured in 77 amniotic fluid samples and in 57 maternal plasma samples. Gestational age ranged from 16 to 40 weeks. Amniotic fluid FFA decreased from 20-4 +/- 6-0 mumol/l in the second trimester to 17-0 +/- 6-0 mumol/l in the last six weeks of pregnancy. This was almost entirely accounted for by a decrease in the concentration of free stearic acid, while oleic acid increased slightly. No correlation between amniotic fluid FFA and maternal plasma FFA was found in samples obtained on the same day. It is suggested that amniotic fluid FFA may partly derive from renal excretion by the fetus.


The influence of pyridoxine on prolactin secretion and milk production in women. Pyridoxine treatment did not inhibit milk secretion in 14 puerperal women. In contrast, bromocriptine was antilactogenic. Pyridoxine appeared not to suppress prolactin secretion.


Puerperal ultrasonic hysterography in the diagnosis of congenital uterine malformations. Puerperal ultrasonic hysterography has been found to be a safe and simple technique. It is suggested as a screening procedure prior to radiological hysterography in patients whose recent reproductive performance suggests a diagnosis of congenital malformation of the uterus.


Tumour associated immunity and immunocompetence in ovarian cancer. Cell-free extracts of autologous ovarian cancer cells evoked a delayed hypersensitivity response (DHR) in skin in four out of six patients in remission from disease and none of seven patients in relapse. These initial results appear to be consistent with in vitro blastogenic assay using the same autologous extracts. To assess whether diminished tumour-associated immunity was due to impaired cell-mediated immunocompetence, blastogenic responses to purified protein derivative of tuberculin (PPD), a secondary recall antigen, and phytohaemagglutinin (PHA) were assessed. Impaired responses to PHA were seen in the relapse group of patients, but responses to PPD did not appear to be impaired in this group.


Lipogranuloma of the uterine parametrium. A case of lipogranuloma of the uterine parametrium is described. The granuloma may have developed following injury to fat cells after a cone biopsy of the cervix or may have resulted from a reaction to paraffin.


Morphology of posterior segment lesions of the eye in patients with onchocerciasis. Posterior segment ocular lesions were examined in 244 selected patients with onchocerciasis from the Sudan-savanna and rain-forest areas of the United Cameroon Republic. Fluorescein fundus angiography was performed on 210 cases. As a result of this study the following conclusions were drawn: 1. The heterogeneity of single lesions, and the occurrence of different types of lesion in a single eye or two eyes, prevented subdivision of the disease into distinct forms and suggested that inflammation alone was responsible for fundus lesions in onchocerciasis. 2. Optic nerve disease, alone or in the presence of choroido-retinal changes, was responsible for a large proportion of the blindness due to posterior segment lesions in onchocerciasis (87.6 per cent in this series).


Acquired arterial macroaneurysms of the retina. Fifteen patients with acquired arterial macroaneurysms of the retina, and an additional woman patient with some 18 aneurysms of the temporal retinal arterial tree of one eye, are described. Acquired aneurysms occur in patients in their sixth decade and older, are usually unilateral, and occur on the major arterial branches posterior to the equator. Customarily only one or two aneurysms are present. Most patients have a history of poorly controlled systemic hypertension or a raised blood pressure at the time of discovery of the macroaneurysm. Focal embolic damage to arterial walls should be considered in the investigation of these patients. In patients with decreased vision from macular oedema and circinate retinopathy, photocoagulation of the macroaneurysm may hasten the improvement in visual acuity.


Angioid streaks and sickle haemoglobinopathies. Five patients had angioid streaks associated with sickle cell haemoglobinopathy. Other diseases associated with angioid streaks were ruled out, as was elastic tissue degenegation in sickle cell patients. After studying over 350 patients, we believe the incidence of angioid streaks in sickle cell disease to be between 1 and 2 per cent.


Reticular tapeto-retinal dystrophy. As a possible late stage of Sjögren's reticular dystrophy. Findings from two patients with a reticular tapetoretinal dystrophy strongly suggested advanced stages of Sjögren's reticular dystrophy. These observations emphasize that, although initially a benign disease, advanced stages of Sjörgren's dystrophy may eventually manifest diffuse photoreceptor and retinal pigment epithelial disease.


Vitreous opacities in primary amyloid disease. A clinical, histochemical, and ultrastructural report. Three patients who initially presented to the ophthalmologist with a history of gradual deterioration of visual acuity showed, on biomicroscopic examination, a linear meshwork of opacities in the vitreous attached to the posterior surface of the lens in the form of pseudopodia lentis. Amyloidosis of the vitreous associated with systemic amyloid disease was diagnosed because of the characteristic nature of the opacities. This was confirmed initially in two of the cases by light and electron microscopical examination of a biopsy of the clinically normal conjunctiva, subsequently by systemic investigations, and finally, by direct histochemical and elecron microscopical examination of the vitreous which became available after vitrectomy in all three cases. It was not possible to examine other members of the families. The relevant literature on vitreous amyloidosis is reviewed in relation to our own findings and the importance of a diagnostic conjunctival biopsy is emphasized. This paper is the first British report on primary vitreous amyloidosis. The pathogenesis of vitreous amyloid opacities is discussed in the light of current concepts of amyloidosis. Since the vitreous opacities due to amyloid deposits appear to be one of the manifestations of systemic amyloid disease, surgical removal of the opacities by vitrectomy may not produce longlasting results. Moveover, in contemplating surgical treatment the ophthalmologist should bear in mind the poor prognosis of systemic amyloidosis.


Optic neuritis in the Landry-Guillain-Barré-Strohl syndrome. A patient with typical Landry-Guillain-Barré-Strohl syndrome (LG-BS) developed bilateral optic neuritis. Laboratory studies showed hypersensitivity to both central and peripheral nervous tissue myelin. The occurrence of optic neuritis is presumably due to autohypersensitivity to central nervous tissue myelin. The initial lesions of the LG-BS syndrome in the peripheral nerves might have liberated sequestered antigens that cross-reacted with central nervous system myelin.


Uhthoff's syndrome.

Uhthoff's syndrome. A total of 125 patients with a previous episode of optic neuritis was followed to assess the subsequent prevalence of transient visual blurring. 32.8 per cent developed this symptom and the precipitating factors were identified. No correlations were found between the development of the symptom and the parameters of optic nerve function studied.


Quantitative tear lysozyme assay in units of activity per microlitre. An accurate method of assaying the tear lysozyme concentration in units of activity/mul has been developed. Normal physiological levels which vary with age have been determined. It has been demonstrated that measurement of the volume of tear fluid collected and assay with a calibrated standard are essential for the accurate determination of the precise concentration of lysozyme in the tear fluid. Tear lyozyme concentration can be used as an index of lacrimal gland function, and in the diagnosis of the sicca syndrome and associated conditions. We believe that this method of tear lyozyme assay has important applications in drug toxicity states in humans (for example, practolol toxicity) and also in animal drug toxicity studies.


A clinical evaluation of the applanation pneumatonograph. The applanation pneumatonograph combines pneumatic and electronic systems. It is used for measuring intraocular pressure, aqueous humour dynamics, and ocular pulsation. In the present study the PTG and the Goldmann tonometer have been used to measure the intraocular pressure in 40 normal and 40 glaucomatous eyes. It has been found that the results of the two instruments correlate well. The PTG readings have, generally, been found to be higher with a mean difference of 0-75 mmHg in the normal and 1-25 mmHg in the glaucomatous eye. The mean ocular pulse as determined in normal eyes was 3-25 +/-0-21 mmHg.


Provocative tests in closed-angle glaucoma. Altogether 119 eyes at risk of developing closed-angle glaucoma were provoked with simultaneous pilocarpine and phenylephrine; of these 74 developed closed-angle glaucoma. The remaining 45 eyes were re-provoked with tropicamide and a further nine developed closed-angle glaucoma. The 36 eyes in which all tests were negative were given no treatment and have been observed for a period of 1 to 7 years (mean 3 years). One has developed closed-angle glaucoma. A scheme for provoking eyes at risk of developing closed-angle glaucoma is described.


The syndrome of closed-angle glaucoma.

The syndrome of closed-angle glaucoma. Closed-angle glaucoma is the result of two mechanisms acting either separately or in combination: 1. Pupil block creates a greater pressure in the posterior than in the anterior chamber, pushing the iris on to the cornea. 2. Increased trabecular meshwork outflow in the presence of pupil block creates a lower pressure in the anterior chamber than in the posterior, pulling the iris on to the cornea. Three main groups of eyes manifest closed-angle glaucoma: (i) Mechanism (I) is necessary and sufficient, but (2) precludes the development of angle closure. (ii) Mechanism (I) is necessary but insufficient. Mechanism (2) must also be present. (iii) Mechanism (I) is necessary and sufficient but (I) and (2) may combine to produce an acute attack.


Intraocular surgery in advanced glaucoma. Loss of visual field subserving macular function has been found to be rare after intraocular surgery in advanced glaucoma. Abrupt changes in refractive error, lens opacity, and suppression must be carefully excluded before macular fixation is deemed lost. Out of 69 intraocular procedures (46 drainage operations, 13 cataract extractons) two patients lost central visual field subserving macular fixation possibly as a result of surgery. There is an increased incidence of postoperative maculopathy in late glaucoma. Three drainage operations out of 46 and four cataract extractions out of 13 had this complication. Altogether 30% of drainage procedures induced deterioration of visual acuity of lenticular origin to a level normally requiring cataract extraction. Trabeculectomy was found to be less likely to cause this sequela than the Scheie procedure at the 6 month postoperative stage. However, 77% of patients with advanced glaucoma and cataract obtained a satisfactory improvement in visual status after cataract extraction.


Clinical assessment of rhodopsin in the eye. Using a standard fundus camera and a photographic technique. A technique based on the method of differential fundus reflectometry is used to assess the availability of rhodopsin in the eye. A defined part of the dark adapted fundus is bleached by a short intense flash of light. The fundus is subsequently photographed in order to record the flux reflected from the bleached area, the optogram, and the surrounding unbleached region. This procedure requires only a few simple modifications to a Zeiss fundus camera before it can be used routinely in the clinic.


Plasma amino-acids in hereditary retinal disease. Ornithine, lysine, and taurine. Plasma free amino-acids were measured in 41 patients with hereditary chorio-retinal degenerations including 26 with retinitis pigmentosa and five with gyrate atrophy of the choroid, six relatives of patients with gyrate atrophy, and 13 normal subjects. Patients with gyrate atrophy had very increased levels of ornithine and slightly decreased mean lysine values. Most relatives had slightly increased ornithine. Taurine, known to be deficient in the plasma of casein-fed cats with photoreceptor degeneration, was normal in all patients. Amino-acid precursors and metabolites of ornithine and taurine were also normal in the plasma. Although the association of high ornithine and gyrate atrophy appears constant, high levels of ornithine alone do not necessarily lead to this degeneration; one patient with known hyperammonaemia, homocitrullinuria and a tenfold increase in plasma ornithine was found to have a normal fundus appearance and normal electroretinogram.


Retinoblastoma occurring in four successive generations. Retinoblastoma occurring in four successive generations is described, followed by a brief discussion on the mode of inheritance of these tumours.


Nocturnal lagophthalmos and recurrent erosion. The symptoms and corneal changes caused by sleeping with one or both eyes open are described in 102 patients. The clinical picture is identical to that of the microform recurrent erosion. The close relationship between the micro- and macro-forms of recurrent corneal erosion suggests that the latter condition is also precipitated by nocturnal lagophthalmos.


Scleritis and episcleritis.

Scleritis and episcleritis. The data from 159 patients (217 eyes) with episscleritis and 207 patients (301 eyes) with scleritis have been investigated in detail and the results analysed with the help of a computer. Of these patients, 91 per cent were followed-up during a period of one to eight years. A new classification is presented which is as follows: Episcleritis (217 eyes) Simple episcleritis (170 eyes) Nodular episcleritis (47 eyes) Scleritis (301 eyes) Diffuse anterior scleritis (119 eyes) Nodular anterior scleritis (134 eyes) Necrotizing scleritis (42 eyes). Of these, 13 were regarded as scleromalacia perforans. Posterior scleritis (6 eyes) The diagnosis is based on an exact clinical examination which is fully described. Episcleritis has been shown to be a benign recurring condition, a mild keratitis being the only occasional complication. Episcleritis does not progress to scleritis, except in the case of herpes zoster which sometimes starts as an episcleritis with the vesicular stage of the eruption, to reappear three months later as a scleritis in the same site. No clear conclusions could be drawn as to the aetiology of episcleritis.


Evaluation of eyes enucleated for scleritis. A series of 30 enucleated eyes, all of which had a primary histological diagnosis of scleritis, was analysed. The average age of patients at enucleation was 68 years; many of these patients had had the disease for more than 30 years. In 40 per cent the diagnosis of scleritis was unsuspected and was often masked by multiple complications. Scleritis with uveitis and glaucoma was the most common combination to come to enucleation. In 82 per cent, pain was the reason for enucleation, which suggests that some patients were on inadequate levels of steroid treatment or were unable to tolerate them. In the series, 37 per cent of the eyes perforated. Perforation occurred in those patients who were having steriods but not in those who were not, but the data are inconclusive as to which method of steroid administration was most likely to cause ocular perforation.


The effects of dietary histidine, methionine and homocystine on vitamin B12 and folate levels in rat liver. 1. L-histidine (20 g/kg) added to vitamin B12-deficient and cyanocobalamin-supplemented diets based on soya-bean flour reduced the growth of rats given the vitamin B12-deficient diet but stimulated growth of rats given the cyanocobalamin-supplemented diet. Liver weight (g/kg body-weight)increased, but the protein content of the livers decreased, in rats given histidine supplements. The histidine was associated with significantly higher folate concentrations in the livers of cyanocobalamin-supplemented rats.


The utilization of diets containing acetate salts by growing lambs as measured by comparative slaughter and respiration calorimetry, together with rumen fermentation. 1. In a comparative slaughter experiment, growing lambs were given concentrate diets in which 14 or 19% metabolizable energy (ME) provided by barley was replaced by sodium, calcium and potassium salts of acetic acid. As the proportion of ME as acetate was increased, energy retention decreased. ME intake was 9271, 9430, and 9217 +/- 67 kJ/d and energy retention was 2698, 2422 and 2280 +/- 71 kJ/d for the diets containong 0, 14 or 19% ME as acetate respectively. There were no differences in protein deposition. The efficiency of utilization of acetate for energy retention (kf) was calculated by difference to be 3 and 10 +/- 13% respectively for the diets containing 14 and 19% ME as acetate.


Mechanisms of heat damage in proteins. 7. The significance lysine-containing isopeptides and of lanthionine in heated proteins. 1. Studies have been made with solvent-extracted chicken muscle, bovine plasma albumin (BPA) and other proteins, all severely heated in the absence of carbohydrates so as to cause a large decrease in their fluorodinitrobenzene (FDNB)-reactive lysine contents. 2. epsilon-N-(beta-L-aspartyl)-L-lysine and epsilon-N-(gamma-L-glutamyl)-L-lysine isopeptides were determined after enzymic digestion of heated chicken muscle, and their content was found to increase as the material was subjected to more heat treatment. Heated chicken muscle was not found to contain lanthionine. Heated BPA, on the other hand, was found to contain lanthionine but not the isopeptides. Both lanthionine and isopeptide cross-linkages were detected in most of the other heated proteins. There was some difficulty in quantifying the amounts of isopeptides formed on heat treatment, because the enzymic digestion procedure used in their isolation appeared to be incomplete. Neither lysinoalanine nor ornithinoalanine was detected in any of the test materials.


The effect of feeding on the motility of the stomach and small intestine in the pig. 1. Electromyographical studies were made on the motility of the stomach and small intestine of five pigs when given a standard diet either ad lib. or as one or two meals/d. Recordings were made from electrodes placed chronically on the antrum, the duodenum and the jejuno-ileum, and a continuous 24 h recording was made every 2d for a period of 4 weeks. A simple "T"-shaped cannula was positioned in the proximal duodenum in two of the pigs.


Light-induced glutamate transport in Halobacterium halobium envelope vesicles. I. Kinetics of the light-dependent and the sodium-gradient-dependent uptake. During illumination Halobacterium halobium cell envelope vesicles accumulate [3H]glutamate by an apparently unidirectional transport system. The driving force for the active transport originates from the light-dependent translocation of protons by bacteriorhodopsin and is due to a transmembrane electrical potential rather than a pH difference. Transport of glutamate against high concentration gradients is also achieved in the dark, with high outside/inside Na+ gradients. Transport in both cases proceeds with similar kinetics and shows a requirement for Na+ on the outside and for K+ on the inside of the vesicles. The unidirectional nature of glutamate transport seems to be due to the low permeability of the membranes to the anionic glutamate, and to the differential cation requirement of the carrier on the two sides of the membrane for substrate translocation. Thus, glutamate gradients can be collapsed in the dark either by lowering the intravesicle pH (with nigericin, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone plus valinomycin), or by reversing the cation balance across the membranes, i.e., providing NaCl on the inside and KCl on the outside of the vesicles. In contrast to the case of light-dependent glutamate transport, the initial rates of Na+-gradient-dependent transport are not depressed when an opposing diffusion potential is introduced by adding the membrane-permeant cation, triphenylmethylphosphonium bromide. Therefore, it appears that, although the electrical potential must be the primary source of energy for the light-dependent transport, the translocation step itself is electrically neutral.


Molecular flow resonance Raman effect from retinal and rhodopsin. We have performed resonance enhanced Raman measurements of retinal isomers in solution (all-trans, 11-cis, 9-cis, and 13-cis) and cetyltrimethylammonium bromide (CTAB) detergent extracts of bovine rhodopsin near physiological temperatures (17 degrees C). In order to measure these photolabile systems, we have developed a general technique which allows Raman measurements of any photosensitive material. This technique involves imposing a molecular velocity transverse to the Raman exciting laser beam sufficient to ensure that any given molecule moves through the beam so that it has little probability of absorbing a photon. We have also measured the resonance Raman spectra of crystals of the same retinal isomers. The data show that each isomer has a distinct and characteristic Raman spectra and that the spectrum of 11-cis-retinal is quite similar but not identical with that of rhodopsin and similarly for 9-cis-retinal compared with isorhodopsin. In agreement with previous work, the Raman data demonstrate that retinal and opsin are joined by a protonated Schiff base. Due to the fact that the Raman spectra of 11-cis-retinal (solution) and rhodopsin show bands near 998 and 1018 cm(-1), a spectral region previously assigned to C-Me stretching motions, it is suggested that 11-cis-retinal in solution is compased of a mixture of 12-s-trans and 12-s-cis, and that the conformation of rhodopsin is (perhaps distorted) 12-s-trans.


Primary structure and microheterogeneities of rat chloroleukemia histone H2A (histone ALK, IIbl or F2a2). Rat chloroleukemia histone H2A, obtained from the F2a2 fraction, has been eluted in two peaks from a Biorex 70 column. The amino acid sequence of rat chloroleukemia histone H2A has been determined and compared to that of calf-thymus histone H2A. The structural studies performed on the tryptic peptides from the maleylated histone and on the thermolysin peptides from the native histone clearly demonstrate the existence of three molecular species of histone H2A depending on the nature of the amino acid residue at positions 16 and 99: H2A-alpha (Ser-16 and Lys-99) accounts for 60% and H2A-betaI (Thr-16 and Arg-99) and H2A-betaII (Ser-16 and Arg-99) for 20% each. A threonine residue at position 16 and a lysine residue at position 99 have been found in calf-thymus histone H2A.


Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro. Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of mercury in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of copper in both fractions. Fractionation of nuclei following in vivo challenge with copper showed enrichment of copper in heterochromatin, relative to euchromatin, while in vivo exposure to mercury resulted in a 20-fold accumulation of mercury in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward mercury and copper similar to that observed in the in vivo metal challenge experiments. The level of mercury which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 mug of Hg/mg of DNA) to that of in vivo euchromatin-bound mercury after 1 month of challenge with dietary metal. In contrast, copper showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both mercury and copper appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of copper.


Interactions between model proteins and deoxyribonucleic acids. Interactions between DNA and model proteins, poly(L-Lys(m)L-Ala(n)), where m + n = 100%, have been investigated using thermal denaturation and circular dichroism (CD). All complexes of DNA with these proteins precipitate in a small range of input ratios, protein to DNA, with the midpoints of all precipitation curves close to a 1:1 ratio of lysine to phosphate. The melting temperature of model protein-bound DNA regions decreases slightly as the alanine content of the model protein is increased, which can be explained as a result of insufficient charge neutralization of phosphates by lysine residues in the model proteins. In the free state, these model proteins possess varying amounts of alpha helix, random coil, or a mixture of these two, depending upon the relative lysine/alanine content. When bound to DNA, the CD of the complex shows a substantial increase in alpha-helical structure for those model proteins with 40-60% alanine, while there is no significant change in alpha-helical structure when the percent alanine is either substantially higher or lower (i.e., 81 or 19% alanine). Only those complexes formed with model proteins having 40-60% alanine undergo a drastic transition from a B-type CD to an A-type in the presence of intermediate ionic strength (0.2 M NaCl, for example). Poly(Lys19Ala81)-DNA complexes show a slight transition toward A-type CD at 0.4 M NaCl or higher. Apparently other factors, in addition to alanine and alpha-helical content, must be responsible for this B leads to A transition. At the other extreme of lysine/alanine ratio, with high lysine content, poly(Lys81Ala19) or polylysine, the presence of NaCl produces a B leads to psi transition. The possible significance of these differences in response to the binding of these model proteins is discussed.


Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein. The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule.


A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver. A procedure is described for the preparation of free and bound polysomes from whole homogenates of rat liver tissue. Liver is homogenized in a conventional medium containing glutathione; then after a 12-min centrifugation at 131000g, the free polysomes in the supernatant are saved, while the membrane-bound polysomes in the pellet are suspended in a mixture of ribonuclease inhibitors (cell sap, 250 mM KCl, and glutathione), homogenized in the presence of detergent (Triton X-100), centirfuged for 5 min at 1470g, decanted, and treated with deoxycholate; the polysomes in the two supernatants are harvested by centrifugation through sucrose gradients containing 250 mM KCl and cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and virtually free of ribonuclease, membranous material, glycogen, deoxycholate, completed protein, and cross-contamination. The recovery of polysomes is approximately 95% and the distribution between the free and membrane-bound state is 25 and 75%, respectively. The molecular weight profiles after sodium dodecyl sulfate-acrylamide gel electrophoresis of the polypeptides completed and released by free and bound polysomes in vitro are different, indicating that there are quantitative differences in the synthesis of various size polypeptides between the two polysome classes. The differential centrifugation procedure is rapid and reproducible, requires much less ultracentrifugation than the isopycnic technique, and provides a nearly quantitative means of separating free and bound polysomes.


Kinetics of recombination of the subunits of human chorionic gonadotropin. Effect of subunit concentration. The rate of formation of human chorionic gonadotropin from its alpha and beta subunits has been measured at neutral pH and 37 degrees C as a function of subunit concentration, using the fluorescence probe, 1,8-anilinoaphthalene-sulfonate (ANS), to monitor the reaction. The subunits were prepared by acid dissociation of the intact hormone (pH less than or equal to 2, 37 degrees C). Following neutralization, the rate of appearance of ANS fluorescence was identical with the rate of recovery of receptor binding activity and both of these properties were completely recovered. Kinetic data obtained over a 100-fold range of subunit concentrations (1.5 to 146 muM) were not compatible with a simple second-order reaction scheme, but required at least one additional step. The data were best fit by a model in which the subunits reversibly form an intermediate complex (alpha + beta in equilibrium alphabeta) which then undergoes a conformational rearrangement to form the native structure (alphabeta leads to H). Ultraviolet difference absorption measurements suggest that most of the change in the environment of the tyrosyl residues occurs during this second step.


The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.


Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate] 2-hydroxy-3-butynoate. 2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, O2. Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band (gamma(max) 600 nm) and lack of fluorescence, making it easily distinguished from the subsequently formed inactivated enzyme, which has no long wavelength absorption (gamma(max) 318, 368 nm) and which is strongly fluorescent. Inactivation is also accomplished by reaction of the reduced enzyme with 2-keto-3-butynoate. The absorbance and fluorescence characteristics of the inactivated enzyme are similar to those of a model compound, C(4a), N(5)-propano-bridged FMN bound to apolactate oxidase. That the modified chromophore of the inactivated enzyme is an adduct involving both the C(4a) and N5 positions is further supported by the spectral and fluorescence changes resulting from treatment of the inactivated enzyme with borohydride.


The structure of the covalent flavin adduct formed between lactate oxidase and the suicide substrate 2-hydroxy-3-butynoate. 2-Hydroxy-3-butynoic acid is a suicide substrate for Mycobacterium smegmatis lactate oxidase. Inactivation occurs by covalent modification of enzyme-bound FMN and does not involve labeling of the apoprotein. The spectrum of the enzyme bound adduct suggests that it is a 4a, 5-dihydroflavin derivative. When this adduct is released from the enzyme, a complex mixture of unstable compounds is obtained. When the initially formed enzyme-bound adduct is reduced with NaBH4, a major stable species can be resolved from the enzyme and can be isolated and purified. The structure was established by appropriate isotope substitutions. Fourier transform NMR spectroscopy, chemical reactivity, and synthesis of a model compound. The structure of the isolated adduct is structure II, Scheme II. The structure proposed for the adduct initially formed on the enzyme is structure VII, Scheme II.


The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure. Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC extraction, poly(ethylene glycol) precipitation, and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000-fold relative to the starting plasma. The apparent molecular weight of the purified factor V was 439,000 +/- 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 239,000; alpha-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.


Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose. A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.


Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization. Erythrocyte spectrin, isolated by aqueous extraction of erythrocyte ghosts, may be freed from contaminating membrane lipids and small amounts of other proteins by gel chromatography in 5 or 10 mM deoxycholate. The purified protein, in deoxycholate, is a mixture of monomers and dimers, both highly asymmetric molecules. The hydrodynamic properties of the dimer closely resemble those of muscle myosin, and spectrin and myosin also have similar circular dichroism spectra. The proportion of dimer to monomer in the purified protein varies from one preparation to another, an observation for which there is no simple explanation. In the absence of deoxycholate, spectrin associated beyond the dimer stage, possibly by loose end-to-end aggregation involving hydrophobic forces.


Hydrolysis of tri- and monoacylglycerol by lipoprotein lipase: evidence for a common active site. The relationship between triacylglycerol and monoacylglycerol hydrolyzing activities of purified rat heart lipoprotein lipase was studied using emulsified trioleoylglycerol and micellar or albumin-bound monooleoylglycerol as substrates. The maximal reaction rates obtained with the two substrates were similar (650 and 550 nmol of fatty acid released per min per mg of protein, respectively). Addition of apolipoprotein C-II or serum increased the maximal reaction rate for the trioleolyglycerol hydrolyzing activity about four-fold, but had no effect on the monooleolyglycerol hydrolyzing activity. Hydolysis of the two substrates apparently takes place at the same active site of the enzyme since (1) mutual competitive inhibition between the substrates could be demonstrated; (2) the rate of inactivation of enzymatic activity with the two substrates in 1.2 M NaCl was the same; (3) similar losses of hydrolytic activity with tri- and monooleoylglycerol were observed in the presence of low concentrations of n-butyl (p-nitrophenyl) carbamide; (4) inhibition of both hydrolytic activities by this compound could be prevented by prior exposure of lipoprotein lipase to either substrate.


Effect of bridging the two essential thiols of myosin on its spectral and actin-binding properties. The circular dichroic and fluorescent spectral properties of the myosin head (subfragment I (SFI)) modified by covalently bridging the two essential thiol groups have been examined. CD spectra of SFI with the two thiols linked through reaction with a bifunctional reagent, N, N'- p-phenylenedimaleimide, show enhancement of the 282-nm minimum similar to that observed for the long-lived kinetic intermediate (Mg**MgADP-Pi) formed during the ATP cleavage reaction. No significant perturbation of the CD band at 282 nm is seen on blocking both thiol groups with the monofunctional reagent N-ethylmaleimide. The fluorescence emission maximum also shifts to lower wavelengths following covalent bridging (from 343 to 340 nm), but no change in fluorescent intensity has been detected. Formation of the covalent bridge completely inhibits interaction of the modified protein with F-actin. These results suggest that the local conformational state of the polypeptide chain formed on bridging the two thiol groups exhibits certain similarities with the state produced following binding of MgATP to native myosin.


Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes. Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.


Histone-histone interactions within chromatin. Crosslinking studies using ultraviolet light. Irradiation of either whole cells or chromatin at 280 nm results in the covalent linkage of histones 2A and 2B, presumably at their mutual binding sites. The reaction is specific and proceeds with high yield (about 80%). Irradiation of reconstituted nucleohistone containing only H2A, H2B and DNA also yields the H2A-H2B dimer. The cross-linking event is sensitive to the conformation of the H2A-H2B pair since the histones must be bound to DNA for maximum cross-linking specificity at low ionic strength. However, the histones must first interact with each other before being deposited on the DNA, since separate addition of the histones to the DNA yields no dimer upon irradiation. If irradiation is conducted at 254 nm rather than 280 nm, DNA-histone cross-linking appears to dominate.


Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase. The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with 125I and 59Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of N-ethylmaleimide which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37 degrees C but nearly all that taken up 4 degrees C was released when the cells were subsequently incubated with trypsin plus N-ethylmaleimide, despite the fact that about 80% of the 59Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells. These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37 degrees C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.


Effect of phase transition on the distribution of membrane-associated particles in microsomes. (1) Rat liver microsomes were studied by freeze-fracture electron microscopy. The distribution of membrane-associated particles indicated the right-side-out orientation of microsomal vesicles. Studies at different temperatures were performed. At 30 degrees C membrane-associated particles are randomly distributed on membrane A-faces, while aggregations of particles are observed at 4 degrees C. (2) Aggregation is dependent on the cooling rates. It can be prevented by shock-freezing. (3) Particle aggregation is also prevented by cholesterol, added to the microsomes in equal molar ratio to the microsomal phospholid content. (4) These findings suggest that particle aggregation is caused by a partial freezing-out of phospholipid molecules during the phase transition from the liquid-crystalline to the gel state. (5) The results are discussed with respect to an observed increase in activation energy of microsomal drug monooxygenation at lower temperature.


Factors affecting the motion of the polar headgroup in phospholipid bilayers. A 31P NMR study of unsonicated phosphatidylcholine liposomes. (1) The 129 MHZ and 36.4 MHZ 31 P NMR spectra of unsonicated liposomes consisting of phosphatidylcholines of varying chain length and unsaturation have been investigated. (2) In the liquid crystalline state the 31 P NMR liposome spectra are similar for both saturated and unsaturated phosphatidylcholines, demonstrating that the motion of the polar headgroup is not sensitive to the fatty acid composition in the disordered liquid crystalline state. (3) Below the hydrocarbon phase transition temperature there is a marked increase in the linewidth of the 31P NMR liposome spectra, indicating a reduction in the motion of the polar headgroup. (4) The addition of equimolar concentrations of cholesterol to phosphatidylcholine eliminates phase transition effects experienced by the polar headgroup. The motion of the polar headgroup is then very similar to that obtained in the liquid crystalline state for pure phosphatidylcholine bilayers. (5) In the liquid crystalline state the motion of the polar headgroup in the phosphate region is insensitive to changes in the available area per phosphatidy-choline molecule.


Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents. Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.


Effect of ethyl acetate on the transport of sodium and glucose in the hamster small intestine in vitro. The effect of ethyl acetate on Na+, water and glucose transport, as well as on glucose and electrolyte intracellular concentrations in everted and cannulated sacs of hamster jejunum, have been studied. Ethyl acetate, a substance that easily penetrates and delivers energy to the cell, strongly stimulates net glucose and Na+ transport. The explanation of the experimental results takes into account the possibility of the existence of an active extrusion of glucose at the level of the basolateral membrane of the enterocyte.


Regulatory characteristics of amino acid transport in newborn rat renal cortex cells. The uptaken of alpha-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 degrees C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na+ during the preliminary incubation. Cycloheximide (50 muM) and purimycin (1 mM) as well as alpha-aminoisobutyric acid, glycine and proline (5 mM) in the preincubation buffer also abolish the effect, while actinomycin D (0.8 muM) partially the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in effux. There is no alteration in the apparent transport Km but an increase in the V for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.


Tryptophan fluorescence and homology in lysozymes and alpha-lactalbumins. Steady-state and transient fluorescence properties of turkey lysozyme, human lysozyme, and bovine alpha-lactalbumin are compared to those of hen lysozyme and hen lysozyme derivatives. Tryptophan fluorescence appears to be sufficiently sensitive to environment so that a small divergence in sequence can alter the fluorescence properties of one or more of the sequentially equivalent tryptophans in these proteins. Thus, an understanding of the fluorescence of one protein cannot necessarily be extended to a homologous protein in a simple manner, and the use of fluorescence to document structural similarity in homologous proteins is likely to be difficult. In addition, the results with alpha-lactalbumin and human lysozyme suggest that multiple conformations of these proteins exist in solution, and that interconversion of these conformations is slow compared to the fluorescence lifetime.


Thermodynamics of the isothermal denaturation of lysozyme by guanidinium chloride. A thermodynamic analysis of the isothermal denaturation of lysozyme by guanidinium chloride has been performed. The analysis is based on the equation which relates the equilibrium constant for denaturation to the preferential binding of denaturant. The equation has been derived previously by thermodynamic methods, whereas in this article a derivation based on statistical mechanics is given. By application of the equation the free energy of denaturation is first calculated and from it, by subtracting the calorimetrically-determined enthalpy of denaturation, the entropy of denaturation is determined.


Preparation of the proteolipid apoprotein from bovine heart, liver and kidney. Proteolipid apoproteins have been prepared from heart, kidney, and liver by dialysis in chloroform/methanol against chloroform/methanol, acidified chloroform/methanol, and chloroform/methanol in succession. They are free of lipids (less than 0.05% P; less than 0.1% carbohydrate). They show a high content of non-polar amino acids, methionine, and tryptophan and contain little or no half-cystine. The differ from neural proteolipid apoproteins by absence of half-cystine, and of covalently bound fatty acids. As recovered from chloroform/methanol solutions, they are soluble in chloroform/methanol and insoluble in water, but a water-soluble form can be prepared by changing the solvent from chloroform/methanol to water in a stream of nitrogen. The chloroform-methanol-soluble form and the water-soluble form are interconvertible. ORD and CD spectra of all proteolipid apoproteins indicate 60-70% alpha-helix content in chloroform/methanol solution and 20-30% alpha-helix in water solution. Sodium dodecyl sulfate gel electrophoresis resolves proteolipid apoprotein into two major components corresponding to ca. 12 000 and 34 000 daltons. With sodium dodecyl sulfate/urea numerous bands appear, with a major one at 30 000 daltons and 8 to 10, ranging downward to 2500. For comparison, neural proteolipid apoproteins also show numerous bands with a major one at 25 000. The marked chemical and physical similarities among all proteolipid apoproteins studied suggest a common role in membrane structures.


The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C. The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.


The demonstration of asymmetric hemoglobin hybrids by polyacrylamide electrophoresis. A simpler, more economical technique than previously reported, that of conventional polyacrylamide gel electrophoresis alone, is described for the detection of asymmetric hemoglobin hybrids of the forms alphaXalphaYbeta2 and alpha2betaXbetaY when bloods from individuals with alpha and beta chain variants were examined. The presence of alpha chain variant hybrids, never before reported, is further evidence that hybrid formation is a more widespread phenomenon than has previously been thought of. Hybrids were found in artificial mixtures of hemoglobins and more importantly, are also reported here for the first time in bloods of individuals heterozygous for hemoglobin variants. These hybrid tetramers were as stable as the parent hemoglobins when examined under anaerobic conditions. The involvement of HbF in the formation of hybrids of the type alpha2betagamma is reported, and an analysis of the possible role of these as well as alpha2betaAbetaS hybrids in the sickling process is presented.


Iron-binding activity of female-specific serum proteins of rainbow trout (Salmo gairdneri) and chum salmon (Oncorhyncus keta). The differences of serum proteins between mature male and female rainbow trout (Salmo gairdneri) and chum salmon (Oncorhyncus keta) were studied electrophoretically and immunologically. Female-specific serum proteins were seen only in females of both species, in the same region as beta-globulin on cellulose acetate membrane electrophoresis and agarose gel immunoelectrophoresis. One of the female-specific serum proteins bound radioactive iron. This protein was partially purified by precipitation by lowering the ionic strength of the serum. The purified material also showed the iron-binding property.


Disulfide-linked cyanogen bromide peptides of bovine fibrinogen. I. Isolation of peptide F-CB3 and characterization of its single disulfide bond by cleavage with cyanide. A fragment F-CB3 which originates from the alpha-chain constituent of bovine fibrinogen could be liberated by CNBr cleavage and was purified by molecular sieve and ion-exchange chromatography. This fragment had a molecular weight of 36 000 and consisted of a single polypeptide chain which is folded into a loop by a single disulfide bridge. Further cleavage of F-CB3 by cyanide or by 2-nitro-5-thiocyanobenzoic acid gave rise to three fragments, CN1, CN2 and CN3, with molecular weights of 23 000, 8000 and 7000, respectively. With both reagents the yield of cleavage did not exceed 50%. Radioactive labeling and amino acid analysis of the purified fragments indicated the order CN1-CN2-CN3 in intact F-CB3. A shorter and apparently degraded form of F-CB3 was observed in some fibrinogen preparations. The shortening involved a region of about 3000 daltons at the N-terminal site of F-CB3, i.e. in fragment CN1.


The affinity of human, rabbit and bovine thrombins for sepharose-lysine and other conjugates. Human, rabbit and bovine thrombins are shown to possess marked affinities for Sepharose-lysine. Using either Xa-activated crude prothrombins (human, rabbit) or a commercial thrombin sample (bovine), the enzyme was isolated in a single chromatographic step by the affinity medium and preparations of high specific activity were obtained. The relevance of bound-lysine for the affinity of the thrombins was studied using other Sepharose conjugates with structures related to Sepharose-lysine. Using freshly activated prothrombins it was found that human and rabbit thrombin uptake required a conjugate with a spacer chain containing a minimum of four carbon atoms in length which supported a terminal amino group. As the thrombin activity aged, affinity for the terminal amino group decreased but the hydrophobic spacer chain became essential for enzyme binding. The active centre of thrombin was not involved in binding to Sepharose-lysine.


The aromatic residue content of the enzyme rhodanese. The aromatic amino acid composition of the enzyme rhodanese has been redetermined. Previous reports have varied from 5 to 11 tryptophans per 26 alanine residues. The present work has quantitated the aromatic residues by a combination of amino acid analysis, solvent perturbation difference spectroscopy, specific residue modification and direct ultraviolet spectral analysis. These methods indicate that rhodanese contains 10 tyrosines, eight tryptophans and 16 phenylalanines per 26 alanine residues. The results for tyrosine and phenylalanine are in reasonable agreement with previous results.


Spectral studies of the tryptophan exposure in the enzyme rhodanese. Tryptophan exposure in the enzyme rhodanese (thiosulfate:cyanide sulfur-transferase, EC 2.8.1.1.) has been studied by the methods of solvent perturbation difference spectroscopy, fluorescence spectroscopy and fluorescence quenching. The data from all these techniques are consistent with two classes of tryptophan:surface and buried. The surface residues appear to be relatively shielded and in an anionic environment. The buried residues have spectral characteristics suggesting they are in a very apolar environment and have less than the expected contact with the peptide backbone. The solvent perturbation difference spectra indicate further that additional tyrosine as well as tryptophan residues are buried as rhodanese is converted from the free enzyme to the sulfur-substituted enzyme: forms which are obligatory intermediates in the catalytic cycle.


Cyanogen bromide fragments of bovine cervical mucus glycoprotein. Treatment of bovine cervical mucus glycoprotein with cyanogen bromide gives four fractions, with the molecular weights of the three major fractions being 230 000, 130 000, and 35 000. The results indicate that, as for other glycoproteins, there are different regions along the protein core, some of which have a high sugar content and others which contain considerably less carbohydrate; it seems likely that the regions of lower sugar content may be important to intermolecular linkages. The data suggest that the basic unit of the glycoprotein has a molecular weight of 550 000-600 000, with its protein core consisting of approx. 1200 amino acid residues.


Magnetic circular dichroism studies on horseradish peroxidase. Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.


The self-association of L-alpha hydroxyacid oxidase. As the published values for the molecular weight of L-alpha-hydroxyacid oxidase vary from 89 000 to 430 000, it is possible that such variations could be due to a concentration dependence of the molecular weight. The molecular weight of rat L-alpha-hydroxyacid oxidase was studied over a wide range of concentrations, using equilibrium sedimentation and gel exclusion chromatography. The partial specific volumes (0.726 and 0.730 for hydroxyacid oxidase A and hydroxyacid oxidase B, respectively) were calculated from the amino acid compositions, and were used to calculat molecular weights from the equilibrium sedimentation data. The molecular weight at infinite dilution was found to be 150 000 for both the A and B isozymes. Both isozymes exhibit association-dissociation behaviour at low concentrations. The self-association of the hydroxyacid oxidase B isozyme can be described by the relation (see article) where K1,2 = 5.4-10(5) M-1 and K2,4 = 1.7-10(5) M-1. Previously published values of the molecular weight of these isozymes are in accord with the observed concentration dependence.


Studies on the covalent structure of eland pancreatic ribonuclease. Studies on the covalent structure of eland (Taurotragus oryx) pancreatic ribonuclease have been performed on tryptic and thermolysin digests. The first 45 residues have been determined with a Beckman sequencer. From the remaining part of the sequence only those peptides were sequenced that differed in amino acid composition with the corresponding peptide of bovine ribonuclease. Eland pancreatic ribonuclease differs in four positions from bovine pancreatic ribonuclease A, but more differences due to a different state of amidation may be present. The absence of an Asn-X-Thr/Ser sequence in the covalent structure of eland ribonuclease (asparagine 34 has been substituted by aspartic acid) explains the absence of a glycosidated component in eland ribonuclease.


Partial structural analysis of the basic chromosomal protein of rat spermatozoa. An investigation of the primary structure of the basic chromosomal protein of rat spermatozoa by automated Edman degradation and by carboxypeptidase digestion has provided a general structural outline of the entire molecule. The exact or approximate location of virtually all residues other than arginine or cysteine is reported. Of particular interest, because of the occurrence of phosphorylated derivatives of this protein, is the location of three of the four serine residues.


Lactate racemase. Hydroxylamine-dependent 18O exchange of the alpha-hydroxyl of lactic acid. The lactic acid racemase (EC 5.1.2.1) derived from Clostridium butylicum catalyzes the racemization of the alpha-18O label. The proposed alpha-carbonyl intermediate for the enzyme-catalyzed reaction has been previously shown to be trapped as an enzyme-bound oxime in the presence of hydroxylamine. This report demonstrates that the formation of the inactive enzyme-bound oxime, followed by reactivation in the presence of an excess of competing free carbonyl (pyruvic acid) results in a complete loss of the alpha-18O label from an original alpha-18O-labeled lactic acid.


Studies on inosine monophosphate dehydrogenase. An associating-dissociating system. The techniques of polyacrylamide gel electrophoresis, sedimentation velocity and frontal analysis on Sephadex have been used to demonstrate that preparations of IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14) from Aerobacter aerogenes consist of a mixture of molecular weight isomers. Further, it has been shown that dissociation of the higher molecular weight forms is promoted by urea, sodium dodecyl sulphate and dithiothreitol. Under conditions comparable to those used for kinetic analyses, the enzyme has a molecular weight of about 86000 and this is the smallest active species that has been observed. In the absence of a reducing agent, the enzyme undergoes polymerization and is devoid of catalytic activity. From the amino acid composition and peptide map, it appears that the molecule with a molecular weight of 86000 is made up of two identical polypeptide chains.


Trimethylamine dehydrogenase from a methylotrophic bacterium. I. Isolation and steady-state kinetics. 1. The isolation of trimethylamine dehydrogenase (EC 1.5.99.7) from a restricted facultative methylotroph to electrophoretic homogeneity is described. 2. The molecular weight and subunit molecular weights were found to be 146800 for the enzyme by sedimentation equilibrium ultracentrifugation and 70000-80000 for the two non-identical subunits by sodium dodecyl sulphate gel electrophoresis. 3. Initial velocity studies indicate that the enzymatic reaction proceeds by a Ping-Pong mechanism. 4. Further kinetic evidence was obtained by analysis of product inhibition patterns using the alternate substrate diethylamine and the products acetaldehyde and ethylamine as product inhibitors, for the release of ethylamine before the addition of phenazine methosulphate and for the existence of an enzyme-two-carbon unit complex as a stable form of the enzyme. 5. Some properties of the unusual prosthetic group of trimethylamine dehydrogenase and its photodegradation product are described in preliminary form.


Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments. The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.


Preparation and properties of ornithine-oxo-acid aminotransferase of rat kidney. Comparison with the liver enzyme. Ornithine-oxo-acid aminotransferase (EC 2.6.1.13) from rat kidney was prepared as a single homogeneous protein as judged by polyacrylamide gel electrophoresis, ultracentrifuge analysis and double diffusion precipitin test. Content of pyridoxal phosphate, light absorption spectra, circular dicroism spectra, Km values, inhibitors, and electrophoretic mobilities of the proteins after reactions with group modifying reagents were similar for the ornithine-oxo-acid aminotransferases of rat kidney and liver. Rates of reaction with group modifying reagents, stabilities to storage at -15 degrees C, and stabilities to temperatures above 55 degrees C differed significantly for the two enzymes. The liver enzyme contained two more cysteine residues than the kidney enzyme as determined by three different methods. Heating the liver enzyme at 66-67 degrees C at pH 5.9 for 1 h decreased the thiol groups titratable by 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2). Uncer the same conditions titratable thiol groups of the kidney enzyme were not decreased. Amino acid analysis revealed probably significant differences in tyrosine and isoleucine content in addition to cysteine. It was concluded that the primary structures of ornithine-oxo-acid aminotransferases of rat liver and kidney are not fully identical.


Characterization of two endopolygalacturonase isozymes produced by Fusarium oxysporum f. sp. lycopersici. Polygalacturonase (EC 3.2.1.15) produced by Fursarium oxysporum f. sp. lycopersici was purified by chromatography on DEAE-cellulose, CM-cellulose, and hydroxyapatite. The purified enzyme consisted of two electrophoretically distinct "isozymes", that behaved as charge isomers during electrophoresis in several different concentrations of polyacrylamide gel. The two isozymes had similar "endo" modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction, reducing group release, and thin-layer chromatography of oligomeric hydrolysis products. Both isozymes hydrolzyed 5% of the substrate bonds in reaching 50% viscosity reduction. The amino acid compositions of the isozymes were similar and their molecular weights were about 37000 as determined by sedimentation equilibrium. Removal of large amounts of carbohydrate during purification did not affect heat stability of the enzymes. A large proportion of the remaining carbohydrate appeared to be covalently linked to the enzyme protein.


Specificity of acid proteinase A from Aspergillus niger var. macrosporus towards B-chain of performic acid oxidized bovine insulin. 1. A comparative study on the mode of action of two highly purified acid endopeptidases (EC 3.4.-) from Aspergillus niger var. macrosporus, acid proteinase A and B, on the B-chain of performic acid oxidized insulin was performed, putting emphasis on the quantitative analysis of the effects of enzyme A. Acid proteinase A behaved very specifically towards the substrate and hydrolyzed four peptide bonds exclusively: three major sites, where hydrolysis proceeded rapidly and almost completely, Asn3-Gln4, Glu13-Ala14, and Tyr26-Thr27; and a minor one, Gly20-Glu21, at which hydrolysis was much slower. 2. The effects of four protease inhibitors, pepstatin, diazoacetyl-D,L-norleucine methyl ester/Cu(II), di-isopropyl phosphorofluoridate, and 1,2-epoxy-3-(p-nitrophenozy) propane on acid proteinases A and B were studied. Acid proteinase A preparations, treated with the former two inhibitors, were used to establish that the major sites of attack were really affected by enzyme A and not by contaminating proteinase B.


Characterization of agarose-bound trypsin. Agarose-bound trypsin (EC 3.4.21.4) was prepared and its properties were compared with those of soluble trypsin. The bound form of the enzyme was found to be equally available to large and small molecular weight substrates as the soluble form. In addition, the bound form of the enzyme showed the same specificity towards protein substrates as the soluble enzyme. However, the agarose-bound trypsin showed greater stability than the soluble trypsin to denaturing conditions for prolonged period of time.


Phosphatidyl-(N-acyl)-ethanolamine. A lipid component of mammalian epidermis. A lipid present in the granular cells of mammalian epidermis was identified as phosphatidyl-(N-acyl)-ethanolamine. The structure was deduced from the ratio of phosphorus : nitrogen : glycerol : fatty acid esters : total fatty acid (1 : 0.94 : 0.97 : 2.1 : 2.9), from analyses of the products of alkaline and acid hydrolyses and from its infrared spectrum. Conclusive evidence was obtained by a direct comparison of the chromatographic properties, degradation products and infrared spectrum of the isolated lipid with those of synthetic 1,2-dipalmitoyl-sn-glycero-3-phospho-(N-palmitoyl)-ethanolamine. The fatty acids attached to the ethanolamine were predominantly saturated (69% of total) and hexadecanoic acid was the major component (41% of total). Phosphatidyl-(N-acyl)-ethanolamine was hydrolysed by a phospholipase C (Bacillus cereus) to diacylglycerol, inorganic phosphorus and N-acylethanolamine. Evidence for the presence of N-acylethanolamine in granular cells and in stratum corneum suggested that an epidermal phospholipase C may be involved in the catabolism of phosphatidyl-(N-acyl)-ethanolamine.