Role of sympathetics in the response to acute venous occlusion. The effect of venous ligation and subsequent blockade of alpha receptors on hindlimb venous pressure, vascular resistance, oxygen extraction and carbon dioxide removal was evaluated in mongrel dogs with use of constant-flow perfusion of the extremity bed. Perfusion, metatarsal vein, femoral vein, and central vein pressures, and changes in paw and thigh circumference were recorded in mongrel dogs in which the hindlimb was perfused at a constant flow. Arterial, metatarsal venous, and femoral venous blood gases were drawn and analyzed for arteriovenous oxygen and carbon dioxide differences. Measurements were repeated following extensive ligation of veins and after subsequent alpha-receptor blockade with Dibenzyline. Findings indicated that ligation of extremity veins resulted in an active increase in total limb resistance upstream from the ligation, and an increase in volume of the limb. Following alpha-adrenergic blockade with Dibenzyline, limb volume increased and oxygen extraction decreased below control levels. The reflex constriction following ligation of veins protected against the detrimental effect of excessive accumulation of fluid in tissue spaces.


The effects of purified cathepsin D infusions in intact animals. Cathepsin D, a potent acid proteinase, has been implicated in the pathogenesis of circulatory shock. The infusion of purified preparations of cathepsin D into intact animals resulted in significant depressions in core body temperature but failed to reproduce the cardiovascular disturbances seen in shock states.


Acid-base status in diabetic mothers and their infants following general or spinal anesthesia for cesarean section. Acid-base status was studied in 30 diabetic mothers and their infants and in 30 healthy mothers and their babies after general or spinal anesthesia for cesarean section. A normal acid-base state was found for the diabetic subjects following general or spinal anesthesia. However, the infants of diabetic mothers given spinal anesthesia had an average pH of 7.20 and a base excess value of -5.67 mEq/l in umbilical-artery blood at delivery. These values were significantly lower than those observed in the infants of the other groups, where the average pH was between 7.28 and 7.30 and the base excess between -1.87 mEq/l and 1.00 mEq/l. These findings were significantly related to maternal diabetes and maternal hypotension.


Double-blind trial of oral carbuterol in bronchial asthma. A double-blind comparison of 2 and 3 mg oral doses of carbuterol, a beta sympathomimetic drug with preferential selectivity for bronchial smooth muscle, was made with 25 and 35 mg doses of ephedrine sulfate over a six-month period in 26 patients with bronchial asthma. Spirometry was done before and three hours after administration of 3 mg of carbuterol or 35 mg of ephedrine sulfate at intervals of two weeks and one, four and six months. The data suggest a cumulative improvement in pulmonary function status with continued oral carbuterol use over the six-month period. However, the clinical effectiveness of the drug on pulmonary function was not clearly distinguishable from that of ephedrine in the present study.


Newer medications to aid treatment of asthma. The selective beta-2 adrenergic bronchodilator preparations should be considered the medications for the first line of defense in the treatment of the average asthmatic patient. Theophylline and derivatives of theophylline are the second line of defense. Cromolyn sodium, atropine and derivatives, corticosteroids and other selected agents follow, depending on the individual case and the circumstances involved.


[Toxico-pharmacological study of lyophilized sodium nitroprusside (SNP)]. Sodium nitroprusside (SNP) shows a strong hypotensive activity not only in normotensive animals but also in the presence of experimentally induced arterial hypertension. Its use in intravenous perfusion produces the rapid induction of arterial hypotension lasting or only slightly exceeding the time of its administration. Most investigators consider this hypotensive activity related to the fall in peripheral resistance, as is supported by the considerable fall in diastolic blood pressure. However, an effect of SNP on heart function cannot be ruled out. After four weeks' treatment at dosages corresponding to one tenth of the LD 50, no local or general toxicity could be demonstrated.


[Use of sodium nitroprusside in cardiology]. Sodium nitroprusside (SNP) is rarely used in cardiology. It is reserved traditionally for severe episodes of arterial hypertension. Certain states of refractory heart failure represent new indications for use, which implies a double haemodynamic monitoring system: continuous control of systemic blood pressure by intra-arterial catheterization; control of pulmonary pressure and repeated measurements of cardiac output. Prolonged treatment requires continuous biological monitoring of toxicity and careful control of kidney function. As a moderator of blood pressure, SNP is remarkably effective. The hypotensive effect is immediate, readily reversible and generally tachyphylaxis is not observed. The effect of SNP on cardiac work is one of double load reduction: mainly a reduction in afterload or pressure and systemic resistance and a reduction in preload or pressure of ventricular filling. In this respect, SNP can be used effectively for severe cases of heart failure intractable to traditional cardio-stimulatory and diuretic treatments and stemming from diverse causes: acute stage of myocardial infarction, ventricular dilatation, mitral papillary syndrome, heart failure, either subacute or chronic, of various causes. As a rule, the immediate results are positive. Taking the patient off the drug can be difficult and may cause a return to the previous haemodynamic situation.


[Changes in serum and urinary magnesium during and after extracorporeal circulation in cardiac surgery in children]. Magnesium plays an essential role in the processes of myocardial contraction and conduction. In cardiovascular surgery, extracorporeal circulation (ECC) in haemodilution results in various electrolyte distrubances, with invariably hypomagnesaemia. A group of 13 children undergoing open heart surgery was compared with a group of 17 children operated upon under the same conditions but with the addition of magnesium to the filling solution for the extracorporeal circulation. Changes in blood and urinary electrolytes during and after ECC were studied. Hypomagnesaemia was seen in both groups after surgery but the provision of magnesium supplements shortened the duration. In addition, blood phosphate levels remained significantly higher in the group receiving magnesium. The significance of these various findings is discussed and a technique for the provision of magnesium supplements is proposed.


[Action of Trivé 1000 on hemostasis and hematocrit]. The action of lipids on haemostasis has already been shown. The authors study possible changes of haemostasis induced by the use of a substance: Trive 1000 containing 3.8 g of Soya oil and 0.3 g of Soya lecithin per 100 ml. Twenty patient received 1 litre per day over a period of ten days and were compared with 16 controls receiving glucose and amino acides without lipids. Studies of haemostasis included: overall coagulation by the thrombo-elastogram, P.R.P. (platelet rich plasma), estimation of the co-factors of the prothrombin complex, thrombin and reptilase times, platelet count, platelet stickiness using the method of HELLEM, platelet aggregation using the method of Born, plasminogen and plasmin activity by the pH STAT method, and P.D.F. levels. These examinations were carried out on the 1st, 3rd, 5th, 7th and 9th postoperative days. The results in the patients receiving Trive 1000 were as follows: no change in blood coagulation, progressive fall in platelet activity in the sense of hypoaggregability which was significant from the 5th day onwards only. There was no change in fibrinolysis. By contrast, there was anaemia, as determined on the basis of a fall in haematocrit, maximal on the 5th day and attributed by the authors to the haemolytic properties of triglycerides. In conclusion, Trive 1000 had no signifcant influence on haemostasis, apart from platelet hypoaggregability of moderate degree after prolonged treatment and perhaps haemolytic anaemia.


[Study of tracheal pressures transmitted by intubation tube balloons]. In the light of experimental studies on balloons of several intubation tubes, the concepts of occlusive pressure, tracheal pressure and minimum occlusive volume are defined and dissociated. An in vivo study made it possible to determine the importance of the tracheal mucous pulse, the suppression of which could contribute to tracheal stenosis.


[A case of gastric rupture caused by oxygen therapy by the nasal route]. The introduction of a catheter for oxygen therapy by the pernasal route, pushed almost into the esophagus led to a gastric rupture. Discussion of this case and of 8 cases already published in the literature.


[Study of a new anesthetic agent: etomidate (R 26490). Electroencephalographic aspects]. Etomidate or R 26490 is a new hypnotic agent produced by JANSSEN and al. in 1971. The first human experimentation was performed in 1972. The authors used Etomidate for 300 anaesthetics given for neuroradiological investigations. Two protocols were used for study: 25 anaesthetics were given with Etomidate as sole anaesthetic to outline its specific properties; 275 angiographies were performed with a combination of Etomidate and fentanyl. In protocol no. 1, the mean consumption was for 1,85 mg/kg/h, in protocol no. 2, the mean consumption was of 0,9 mg/kg/h. Pharmacological study of this drug defines it as a short acting hypnotic agent of low toxicity. Clinical observation shows cardiovascular and respiratory stability. However, frequent myoclonias were noted, when Etomidate was given as a sole anaesthetic. EEG recording and evoked response encephalography helped to outline some effects of Etomidate on central nervous system.


Replication of swine and equine influenza viruses in canine kidney cells. Both swine and equine influenza viruses were replicated in Madin-Darby canine kidney cells. Hemagglutinin and complement-fixing antigens were synthesized at 4 to 8 hours postinfection and reached maximal synthesis at 16 to 20 hours. Neuraminidase activity increased 9- to 11-fold; 50% of the total hemagglutinin antigen was made before the infective virus progeny was detected.


[Relationship between the quality of the inoculum material and carminomycin biosynthesis by a culture of Actinomadura carminata]. The effect of the inoculum mycelium quality on carminomycin biosynthesis by Actinomadura carminata was studied. The time of the organism growth on the culture medium containing cornsteep liquor continued for 6 hours without losing by the inoculum of its seeding qualities during that period. The mycelium growth in the inoculum was more intensive under conditions of moderate aeration, i.e. 0.98-2.64 mg O2H1-min. Anincrease in the aeration rate up to 18.56 mg O2/1-min resulted in the growth suppression up to 40 per cent. No correlation between the aeration rate during the inoculum growth and the culture capacity for carminomycin biosynthesis and of the content of the complex in active components the fermentation medium were observed, when a 5-10 per cent of inoculum was used.


[Physicochmemical characteristics of the new antineoplastic antibiotic, nocamycin]. Nocamycin is produced by Nocardiopsis syringae. It is recovered from the culture fluid by extraction with chloroform. The molecular weight of the crystalline antibiotic is 503, its melting point is 147--149 degrees, [alpha]20 degrees D = --50 degrees (c. 0.21, chloroform), lambdamax235 and 348 nm(E1%sm= = 150 and 420), the summation formula is C26 H33NO9, the biological activity is 100000 Units/mg with respect to Bacillus mycoides. Nocamycin forms salts with alkalies soluble in water. On hydrolysis with an alkali it forms carbonic acid having no ester bond (IP spectrum) and methoxylic group present in the antibiotic molecule. Nocamycin is a new natural substance.


[Effect of different forms of nitrogen in the biosynthesis of gentamicin by a Micromonospora purpurea var. violacea 1935 culture]. The effect of different inorganic sources of reduced and oxidized nitrogen on biosynthesis of gentamicin was studied. It was shown that both the ammonium and the nitrate nitrogen were consumed by the antibiotic-producing organism. Out of all the salts tested only ammonium sulfate stimulated the biosynthesis of gentamicin. The positive role of this salt was due to both the ammonium and the sulfogroup, since the presence of the sulfogroup alone in sodium sulfat, magnesium or sulfuric acid resulted only in partial stimulation of gentamicin biosynthesis. Simultaneous addition of sulfuric acid ammonium nitrate which suppressed the antibiotic biosynthesis when used alone was equal to introduction of ammonium sulfate.


[Ways to optimize the technological process of oleandomycin reextraction]. Dependence of the oleandomycin distribution coefficient on pH of the acqueous phase and temperature in the system of butylacetate extract-water acidified with orthophosphoric acid was studied. With a purpose of intensification of the process of oleandomycin reextraction, decreasing the antibiotic inactivation and evaporation of the organic solvent it was proposed to perfom oleandomycin extraction at pH 4.0--5.0 accompanied by simultaneous decreasing of the temperature.


Normal and abnormal middle ear ventilation. Studies in infants and children have suggested a functional rather than mechanical obstruction of the Eustachian tube as a predisposing factor in middle ear effusions (MEE). To simulate this condition in the laboratory, an animal model was prepared using juvenile Rhesus monkeys. The tensor veli palatini muscle was transected or expunged posterior to the hamulus of the medial pterygoid lamina. Transection of the muscle resulted in negative middle ear pressure without effusion, whereas when the muscle was expunged, the animals developed a brief episode of negative middle ear pressure followed by a persistent MEE that was sterile for bacteria. An acute bacterial MEE developed following instillation of Streptococcus pneumoniae into the nasopharynx of animals that had had a previous unilateral transection of the muscle. The condition of the middle ear was documented by impedance measurements and presence of the effusion was verified by myringotomy. Animals were periodically examined and tested for Eustachian tube ventilatory function over a period of one year. Before surgical alteration of the tensor muscle. Eustachian tube function tests demonstrated normal ventilatory function, whereas, functional Eustachian tube obstruction patterns similar to studies in children who had MEE were found during the postoperative period. Only after the development of a reliable animal model can current and future methods of management of MEE be tested under controlled laboratory conditions. These data suggest that the Rhesus monkey appears to be an excellent model for the study of normal as well as abnormal tubal function.


[Role of the functional groups of the sibiromycin molecule in DNA binding]. Biological activity of 2 derivatives of sibiromycin, an antibiotic close by its chemical structure to antramycin and their capacity for formation of complexes with DNA was studied. Anhydrosibiromycin like sibiromycin formed a complex with DNA. The antibiotic increased the DNA melting point but to a less extent than sibiromycin. Anhydrosibiromycin had a low activity in the system of DNA-dependent RNA-polymerase. The low biological activity of anhydrosibiromycin must be due to instability of the antibiotic complex with DNA. Methyl ether of sibiromycin by the phenol hydroxyl, the other derivative of sibiromycin had no biological activity and did not interact with DNA. On the basis of experimental data it was suggested that definite functional groups of the sibiromycin participated in DNA binding.


[Antibiotic sensitivity of the bacterial microflora from the sputum of children with acute pneumonia]. Bacteriological analysis of sputum of 598 children with acute pneumonia was performed. Sensivity of 1348 cultures belonging to 8 bacterial species with respect to benzylpenicillin, streptomycin, chloramphenicol, tetracycline, oxytetracycline, chlortetracycline, erythromycin, oleandomycin, neomycin and monomycin was determined. It was shown that the sputum microflora was often resistant to the antibiotics widely used in the medical practice for prolonged periods of time, such as benzylpenicillin, streptomycin, chloramphenicol, tetracyclines. However, it usually remained sunsitive to neomycin, monomycin, erythromycin and oleandomycin. It was found that antibioticogrammes defining the antibiotic choice were of great significance for therapy of acute pneumonia.


Activity of spectinomycin against anaerobes. The in vitro inhibitory activity of spectinomycin was tested against various anaerobic bacteria. Different results were obtained with different media and with different initial pH's of the media. The highest minimum inhibitory concentrations for Bacteroides fragilis ([Formula: see text] 128 mug/ml) were obtained with the use of Wilkins-Chalgren agar (pH 7.2) and Brucella blood agar (pH 7.0). Brucella blood agar at higher pH's (7.4 and 8.0) and Mueller-Hinton and Diagnostic Sensitivity Test agars produced lower minimum inhibitory concentrations (32 and 64 mug/ml). This same relationship between spectinomycin activity and pH of the medium was, in general, observed with these media and other anaerobes, including isolates of B. melaninogenicus, Fusobacterium, gram-positive cocci, Clostridium perfringens, and C. ramosum. The variable results observed in this study and in two others make it difficult to predict the clinical usefulness of spectinomycin in the treatment of anaerobic infections. It is probably most appropriate to be guided by results obtained with Wilkins-Chalgren agar and the method proposed as a reference to the National Committee for Clinical Laboratory Standards. These results indicate that spectinomycin is not a potent inhibitor of B. fragilis or other clinically significant anaerobes.


Differential susceptibility of spleen focus-forming virus and murine leukemia viruses to ansamycin antibiotics. The streptovaricin complex (SvCx) and rifamycin SV derivatives display potent antiviral activity against the polycythemic strain of Friend leukemia virus (FV-P), as measured by a reduction in the number of spleen foci produced in mice. Such reductions may be explained by inactivation of functions of (i) the spleen focus-forming virus (SFFV), (ii) its "helper" murine leukemia virus (MuLV), or (iii) both viruses normally present in FV-P. We noted that preincubation of FV-P with fractionation products of SvCx, or derivatives of rifamycin SV, at low concentrations (3 to 5 mug/ml) reduces the number of spleen foci 80 to 97%, whereas titers of MuLV (from the same inoculum) remain unaffected (MuLV titers were measured by XC, S(+)L(-), and "helper activity" assays). Our findings indicate a remarkable biological selectivity of ansamycins, as well as nonansamycin components of SvCx, against the transforming and defective spleen focus-forming virus as compared to MuLV. Thus, the drugs might be useful in distinguishing other types of oncornaviruses.


Effects of small-particle aerosols of rimantadine and ribavirin on arterial blood pH and gas tensions and lung water content of A2 influenza-infected mice. The respiratory pathophysiology of A2 influenza infection was studied in mice treated with small-particle aerosols (SPA) of rimantadine or ribavirin. Untreated infections in mice resulted in survival rates of 15% or less and were characterized by (i) severe hypoventilation (decreased P(O2) and increased P(CO2)), (ii) compensated respiratory acidosis (increased P(CO2) and HCO(3) (-), with normal pH), (iii) pneumonia with increased ratio of wet/dry lung weight, and (iv) hypothermia. Treatment with SPA of rimantadine (21 mg/kg per day for 4 days) beginning 72 h after virus challenge significantly improved survival rate (80%) but failed to alter lung pathology from that found in infected, untreated mice. Rimantadine treatment decreased somewhat the severity of hypoventilation, respiratory acidosis, lung wet weight, hypothermia, and lung virus titers from that observed in infected, untreated mice. SPA of ribavirin (26 mg/kg per day for 4 days) initiated 6 h after SPA exposure of mice to virus significantly improved survival rate (95%) and reduced lung virus titers and lung pathology. Gas exchange and pulmonary edema in ribavirin-treated, infected mice were significantly improved over those of infected, untreated controls. The mechanisms for increased survival rates induced by SPA of rimantadine remain uncertain, since increased survival rates could not be ascribed entirely to improvements in lung functions. In contrast, however, ribavirin treatment appeared to improve survival rates by reducing major lung pathology and pulmonary dysfunction. This was probably mediated through the antiviral effects of ribavirin.


Delineation of the relative antibacterial activity of cefamandole and cefamandole nafate. By conventional laboratory evaluation procedures, the in vitro antibacterial activities of cefamandole and its O-formyl ester, cefamandole nafate, appear virtually identical. When the activities of these two compounds were examined for their ability to lyse log-phase cultures of susceptible bacteria, however, cefamandole was found to be about 10 times more active than cefamandole nafate. Cefamandole nafate was shown to be rapidly converted to cefamandole in bacteriological media, with a half-life of less than 1 h at a pH of 7.0 or above. At pH 6.0, in log-phase inhibition experiments, however, cefamandole nafate is more stable, allowing delineation of the activity between cefamandole and cefamandole nafate. The efficacy of cefamandole was identical to that of cefamandole nafate in treating experimental animal infections, indicating that rapid conversion of cefamandole nafate to cefamandole occurs in vivo.


Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae. A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.


Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media. Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.


Graft-versus-host reaction. Cutaneous manifestations following bone marrow transplantation. The distinctive cutaneous changes that occur in both the acute and chronic forms of the graft-vs-host reaction (GVHR) are described in two living patients in whom the GVHR developed after bone marrow transplantation for aplastic anemia. In the skin, the mild form of the acute GVHR is recognized as a subtle macular erythema, and the severe form appears as erythematous papules and violaceous macules with scale. Skin biopsy specimens in both of the acute forms show vacuolar alterations of the epidermal basal-cell layer with a perivenular infiltrate of lymphocytes. The chronic GVHR evolves from generalized scaling to diffuse areas of aclerotic and atrophic skin with a curious reticulated hyperpigmentation, ulcerations, and alopecia. Histopathologic study shows collagenization of the dermis that can be correlated with the clinical sclerodermoid changes. Owing to its visibility, the skin offers a unique opportunity for the early recognition of the GVHR.


The salt relations of Dunaliella. Further observations on glycerol production and its regulation. Dunaliella tertiolecta (marine) and D. viridis (halophilic) were each trained by serial transfer to grow at salt concentrations previously regarded as the other's domain. D. viridis then had a salt optimum at 1.0-1.5 M sodium chloride whereas that for D. tertiolecta was less than 0-2 M. Nevertheless D. tertiolecta grew faster than the halophil at all salt concentrations up to 3.5 M, the highest at which they were compared. Both species accumulate glycerol, which is necessary for growth at elevated salinities and which responds in its content to water activity (aw) rather than specifically to salt concentration. Variation in glycerol content is a metabolic process which occurs in the dark from accumulated starch as well as photosynthetically. Regulation of glycerol content by aw does not require protein synthesis. The NADP-specific glycerol dehydrogenase of each of the algae is likely to be directly involved in the regulation of glycerol content. Kinetic studies, together with those described in an earlier publication, show that the enzyme has regulatory properties and that both glycerol and dihydroxyacetone act as effectors as well as reactants. A mechanism of the reaction is tentatively proposed.


[Studies on the thiamine transport system in Bacillus cereus (author's transl)]. The thiamine transport system in Bacillus cereus exhibits rhythmical changes of resorption- and excretion-phases lasting 1-2 h. These main phases are subdivided in shorter ones with an average duration of 45 s. The velocity of the thiamine uptake is influenced by pH, temperature, age of cells, energy and substrate supply and thiamine concentration of the medium. The Michaelis-Menten-Kinetic can be used to describe the uptake: Km = 1.98 x 10(-8) M; Vmax = 1.19 x 10(-6) mol/g dry weight x min. The rate is enhanced by K+, Ca2+ and Mg2+, and inhibited by Pyrithiamin, EDTA, H+-ions, proton donors and proton acceptors; OH(-)-ions cause a change in the direction of transport. A theoretical explanation can be given by assuming a coupling of the thiamine permeation with proton movements in the membrane.


Nitrogen dioxide and pulmonary proteolytic enzymes. Effect on lung tissue and macrophages. Hamsters were exposed to 30 ppm nitrogen dioxide (NO2) for 2 and 50 days and sacrificed. Pulmonary lavage was carried out on a portion of each group to obtain an alveolar macrophage fraction. Proteolytic activity (P.A.), as measured by caseinolysis at pH 3.0 and pH 5.0, increased nearly twofold in the 2-day NO2 lung extracts and fourfold in the 50-day NO2 samples. P.A. in macrophage extract at pH 3.0 increased tenfold with both 2- and 50-day NO2 exposure. Lung extract hydrolysis of specific esterase and amidase substrates and susceptibility to activators and inhibitors of proteolytic enzymes are consistent with the presence of lysosomal cathepsin A, B1, B2, C, D, and E. The lack of NO2-induced increases in P.A. at physiologic values of pH may be the basis of the lack of significant pulmonary tissue destruction observed in rodents exposed to NO2 for 2 and 50 days.


Utilization of nitrogen compounds and ammonia assimilation by Chromatiaceae. Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH4Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina. Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrated in the above organisms grown on NH4Cl.


Substitute and alternative neurotransmitters in neuropsychiatric illness. The accumulation of structural analogs of normal synaptic neurotransmitters (substitute or "false" neurotransmitters) can have profound behavioral and neurologic consequences. Such abnormalities of the metabolism of amines and amino acids may explain behavioral and neurologic changes in hepatic failure. Accumulations of substitute transmitters may mediate other neuropsychiatric phenomena in states of inborn or acquired metabolic error or after certain drugs associated with psychosis. In developing hypotheses concerning relationships between neuropsychiatric disorders and neurotransmitter metabolism, this mechanism might be considered as a novel approach.


[Aminoacid-p-nitroanilides splitting activities in the mature human placenta (author's transl)]. A long experience in the treatment of endometrial cancer shows that the therapeutic measures vary considerably with regard to the extention of the tumor, the age of the patient and her general condition. This circumstances and particularly the critical study of the therapeutical results give the limits, within a certain kind of operation can reasonably be recommended. The intercurrent deaths are an important factor in the statistical evaluation of therapeutical success. As long as the tumor is limited to the body of the uterus, the corrected 5-year recoveries are about 90%. Considering all the circumstances, it becomes obvious, that in this stage, the simple abdominal hysterectomy with removal of the adnexa is still the operation of choice. Beside the histological degree of differentiation, the depth of the muscular invasion is the most important prognostic factor which might lead to additional measures. Postoperative radiation therapy of the vagina reduces considerably the incidence possibility of vaginal apex recurrence. In stage II radical hysterectomy must be considered, although we are aware of the fact, that a simple hysterectomy and bilateral salpingo-oophorectomy combined with radiation treatment may yield just as good results. In the clinical stage III laparotomy ought to be used more frequently. Also in case of operative intervention additional radiotherapy is mostly useful.


[Aminoacid-p-nitroanilides splitting activities in the mouse human placenta (author's transl)]. Proteolytic activities of human placental extract are determined with the aid of a sensitive micromethod, based upon the Bratton-Marshall-reaction, and aminoacid-p-nitroanilides as substrates. The activities are differentiated by using 19 substrates, by adding 10 effectors, and by incubating the assays within the pH-range from 6.0--8.4. Besides Oxitocinase- and Aminopeptidase-like activities, a very active neutral peptidase, so far unknown in human placenta, an acid peptidase, e.g. Cathepsin C or Dipeptide-aminopeptidase II, and some other enzymes, not yet indentified by us, are demonstrated.


Activities of some peptidases and glycolytic enzymes in cells of the amniotic fluid. i. Pregnancies terminated prematurely, at term, and postmaturely. Activities of leucylaminopeptidase, gamma-glutamyl transpeptidase, oxytocinase and aldolase were significantly elevated in the cells of amniotic fluids from cases of premature delivery, compared with cases of delivery at term. The authors conclude that examination of the activities of the aforementioned enzymes can be utilized in prenatal diagnosis of the state of maturity of the fetus.


Variables of the rubella hemagglutination tests employing freeze-dried erythrocytes. The variables which affect the interaction between freeze-dried one-day-old chick erythrocytes and rubella hemagglutinin prepared from rubella-infected porcine kidney cells were defined and evaluated. The sensitivity of the hemagglutination (HA) reaction is much greater at pH 6.0 to 6.2 than at pH 7.0 to 7.5 HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid) diluent with added Ca+ or Mg2+ ion gave four- to eightfold higher HA titers than one without divalent cations. The development of agglutinated and non-agglutinated erythrocyte patterns depended much upon the concentrations of gelatin and albumin in the HEPES diluent. Gelatin especially was essential to obtain stable and clearly distinguishable patterns. Optimal conditions for the agglutination of freeze-dried erythrocytes by rubella hemagglutinin were provided when a HEPES-buffered saline at pH 6.2, containing 10(-3) M CaCl2, 0.2 per cent bovine serum albumin, and 0.0025 per cent gelatin was employed throughout as a diluent for serum, hemagglutinin, and freeze-dried erythrocyte suspension. This diluent gave maximally sensitive and reproducible results in rubella HA and hemagglutination-inhibition (HI) tests employing freeze-dried erythrocytes.


Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases. Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.


Identification and partial characterization of phospholipases in isolated adrenocortical cells. The effects of synacthen [corticotropin-(1--24)-tetracosapeptide] and calcium ions. Phospholipase A activity was determined in homogenates and subcellular fractions of trypsin-dispersed cat adrenocortical cells. At pH 7.4 homogenate phospholipid hydrolysis was activated by added Ca2+ and inhibited by EGTA. Phospholipid degradation in the presence and absence of Synacthen was completely blocked by EGTA. Ca2+-dependent activation of a membrane-bound phospholipase may be a critical control mechanism for regulating the molecular changes taking place during stimulation by Synacthen.


A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus. In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a 'fast cycle' and a 'slow cycle' [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme was considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H2O2 or NaN3 but slightly decreased by KCN. Both H2O2 and the reducing radical anion CO2-- caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55 degrees C), and as temperature increases the slow cycle becomes relatively more important. Arrhenius parameters of the rate contants were estimated. The possible identity of the various forms of the enzyme is considered.


Purification and some properties of arylsulphatases A and B from rabbit kidney cortex. Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg  for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N--acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.


Purification and properties of a cellulase from Aspergillus niger. A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.


Respiratory effects and amnesia after premedication with morphine or lorazepam. Lorazepam, a new benzodiazepine, was compared with morphine for premedication. Ten patients received morphine 10 mg/70 kg i.m. and 10 received lorazepam 4 mg/70 kg i.m. Respiratory effects were assessed from the change in slope (S) and intercept (B) of the carbon dioxide response line, using a development of Read's rebreathing method. Morphine depressed S by 47% (P less than 0.01), but after lorazepam S increased by 27% (P less than 0.05), neither drug altering B significantly. In two volunteers lorazepam was assessed by both the rebreathing and the steady-state methods; after lorazepam S was smaller by the steady-state than by the rebreathing technique. The findings for lorazepam are consistent with the known effects of sleep on carbon dioxide sensitivity. Amnesia lasting 4-8 h occurred in all patients who received lorazepam so that pain and nausea during this period were not recalled, but no patient who received morphine experienced amnesia. We conclude that lorazepam merits further study, particularly where sedation without respiratory depression is needed, as in obstetrics, and where amnesia for uncomfortable procedures is required.


Correlation of biochemical data with Apgar scores at birth and at one minute. A prospective study of 66 unselected neonates revealed a better correlation of umbilical artery blood biochemical data with the Apgar score at birth compared with the score at 1 min. The data confirmed also that inclusion of the score for "colour" detracts from the value of the total score. An Apgar score at birth is more valuable than the score at 1 min.


Cancer of the oral cavity, pharynx/larynx and lung in North Thailand: case-control study and analysis of cigar smoke. The unusually high relative frequency of cancer in the laryngeal region in males (18% of all histologically diagnosed cancers) and a sex ratio of unity for lung cancer in Northern Thailand were further explored in a hospital-based case-control study in Chiang Mai. This compared patients having cancers of the oral cavity (including oropharynx), larynx, hypopharynx and lung, with controls in relation to smoking and chewing habits. Statistical analysis indicated that chewing betel is strongly associated with the occurrence of oral cancer in both sexes, and with cancer of the laryngeal region in males. No factors were strongly linked to lung cancer in men, but, in women, urban residence and miang chewing were associated with lung cancer. Analysis of smoke from the two main types of cigars smoked in the region showed that both had high tar content, but there were marked differences in pH. Smoking cigars with alkaline smoke and high tar had an increased risk for laryngeal cancer in males, whereas other cigars with acid smoke and high tar together with manufactured cigarettes had increased risks for lung cancer. These increased risks were not, however, statistically significant.


Blocking factors against leucocyte-dependent melanoma antibody in the sera of melanoma patients. Previous studies, using plasmapheresis to remove blocking factors of cell-mediated cytotoxicity to melanoma cells from the circulation of melanoma patients, suggested that leucocyte-dependent antibody to melanoma cells may also be blocked by factors in their sera. The present study confirms these findings, by showing that most patients with disseminated melanoma had melanoma LDA activity in the IgG fraction when this was separated from their sera. This also applied to a high percentage of patients with primary melanoma. Evidence that the blocking factors may be immune complexes was shown by experiments in which LDA activity to melanoma cells was revealed after acidification of melanoma sera to dissociate immune complexes, followed by ultrafiltration through membranes retaining molecules of size greater than 100,000 daltons. Blocking of LDA activity in the retentate recurred when the retentate was recombined with the filtrate. Further studies indicated that the blocking activity showed affinity for the target cell and not the effector cell. Preliminary analysis of the specificity of the blocking suggests that this was similar to that of melanoma antisera. These results appear to show that blocking of LDA activity to melanoma cells is common in melanoma patients and that the assay system may provide a quantitative method for their analysis that may yield information of biological importance in the management of melanoma patients.


Serum ferritin in haemochromatosis: changes in the isoferritin composition during venesection therapy. The isoferritin composition of serum ferritin in 13 patients with untreated idiopathic haemochromatosis (IHC) has been shown to differ from normal in exhibiting an increase in isoferritins in the pH range 5.54-5.62. A similar change was observed in four patients with gross iron overload secondary to haemolytic anaemia. During the course of venesection therapy there was a progressive rise in isoferritins of pI 5.02-5.06 relative to the more basic isoferritins. These observations are consistent with previous studies showing alterations in tissue isoferritins in untreated IHC before and after venesection therapy and they are compatible with the hypothesis that the more basic isoferritins correspond to a 'storage' ferritin and the more acidic to a 'secretory' ferritin. The studies also provide further evidence for a possible biological role of the individual isoferritins.


Decreased deformability of erythrocytes in haemolytic anaemia associated with glucosephosphate isomerase deficiency. Deformability of erythrocytes from four patients with different types of glucosephosphate isomerase (D-glucose-6-phosphate ketoisomerase, GPI) deficiency has been determined by cell filtration. Young as well as whole erythrocyte populations had a markedly increased rigidity and an abnormally strong attachment of haemoglobin to the inner surface of isolated membranes. Acidic environment may enhance membran rigidity in vitro and also during passage of the erythrocytes through the spleen. The decrease of deformability at a pH of 6.8 was most pronounced in the splenectomized patients, and likewise in erythrocytes from the splenic artery, which were obtained from one patient during splenectomy. It is suggested that the metabolic environment of the spleen, with its low pH, impairs the deformability of GPI-deficient erythrocytes and predisposes them to splenic sequestration. The clinical improvement of all patients following splenectomy which is accompanied by an increase of the erythrocyte survival time and by unchanged reticulocyte counts, is in accordance with this view.


A to O bone marrow transplantation in severe aplastic anaemia: dynamics of blood group conversion and demonstration of early dyserythropoiesis in the engrafted marrow. A to O bone marrow transplantation was performed in a 25-year-old male affected with severe aplastic anaemia, the donor being an HLA compatible brother. Three plasma exchanges had to be performed with an Aminco separator to remove the original and recurring anti-A isohaemagglutinins. The dynamics of O to A blood group conversion were followed by means of differential agglutination. An early wave of marked dyserythropoiesis was observed in the engrafted marrow. Mild to moderate GvHD was treated successfully with MTX, bolus high dosage 6-methylprednisolone and, at relapse, with intravenous ALG.


Reciprocal interaction of hemoglobin with oxygen and protons. The influence of allosteric polyanions. The interaction of three inositol esters, inositol hexaphosphate (IHP), inositol pentaphosphate (IPP), and inositol hexasulfate (IHS), with hemoglobin has been investigated. The proton uptake method was used to obtain the six binding constants for deoxy- and oxyhemoglobin. These data combined with oxygen binding curves over a range of cofactor concentrations were used to test theoretical and empirical equations relating the affinity of hemoglobin for oxygen and allosteric effectors. The Bohr and Haldane coefficients in the presence of the inositol esters are unequal at low, but not at high, concentration of the cofactors. The maximum value reached by both parameters increases with the number of negative charges of the polyanion. 2,3-Diphosphoglycerate (DPG) differs sharply from the inositol esters since even at high concentrations of this cofactor, the Haldane coefficient remains elevated. This is a reflection of the negligible affinity of DPG for fully oxygenated hemoglobin.


Conformations of synthetic tetradecapeptide renin substrate and of angiotensin I in aqueous solution. The properties of aqueous solutions of synthetic renin substrate tetradecapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) were examined through electrometric titrations, infrared and circular dichroism spectroscopy, and spectrofluorometry. Titration studies of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) were also made, whose results indicated a flexible folded conformation similar to that previously proposed for the octapeptide angiotensin II, with a possible additional beta turn at the C terminus. The experimental results of the tetradecapeptide study, associated with Chou and Fasman calculations and with an analysis of structure-activity relationships in renin substrates and competitive inhibitors, led to the proposal of a beta turn involving the His-Pro-Phe-His sequence of the tetradecapeptide. This beta turn would be stabilized by beta-antiparallel interaction between residues 3-4 and 10-12 and by electrostatic attraction between the N-terminal ammonium and C-terminal carboxylate groups and would be destabilized below pH 5 by electrostatic repulsion between His6 and His9. The capacity to assume this conformation is related to structural requirements for renin substrates and competitive inhibitors.


Effects of quaternary ligands on the inhibition of acetylcholinesterase by arsenite. Arsenite inhibits acetylcholinesterase in a second-order reaction. The rate and equilibrium constants depend upon pH and have values on the order of 10(2) M-1 min-1 and 10(5) M (dissociation), respectively. Some quaternary ammonium ligands completely block the arsenite inhibition of the enzyme, others decrease the rate of the reaction and some, notably pyridine-2 aldoxime methiodide, greatly accelerate the rate of the reaction, up to 220-fold. Accelerators may bind at a separate enzyme site distinct form the anionic site involved in substrate binding. Although the kinetic data are consistent with a covalent reaction between arsenite and acetylcholinesterase, chemical evidence excludes the involvement of sulfhydryl groups which are usually implicated in arsenite inhibition.


Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5. Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.


Phosphoglycerate mutase from wheat germ: studies with isotopically labeled 3-phospho-D-glycerates showing that the catalyzed reaction is intramolecular. Appendix: phosphoglycerate mutase from wheat germ: isolation, crystallization, and properties. The isomerization of 3-phospho-D-glycerate and 2-phospho-D-glycerate catalyzed by the cofactor-independent phosphoglycerate mutase from wheat germ (the isolation and crystallization of which is described in the Appendix) has been shown to be intramolecular by two methods. Mass-spectrometric analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[18O]phospho-D-glycerate shows that there is no exchange of labeled phosphoryl group between carbon skeletons in the mutase-catalyzed reaction. Analysis of the products from the isomerization of a mixture of 3-phospho-D-[2(-2)H]glycerate and 3-[32p]phospho-D-glycerate by a method involving the kinetic discrimination between 2(-2)H and 2(-1)H species using the enolase isotope effect similarly shows that the wheat germ phosphoglycerate mutase mediates an intramolecular transfer of the phosphoryl group.


Preparation and properties of a new DNase from Aspergillus oryzae. A DNase present in commercial preparations of Aspergillus oryzae alpha-amylase was purified 1550-fold in 25% yield by acetone precipitation and by chromatography on diethylaminoethyl- and carboxymethylcellulose. The enzyme was isolated free of contaminating RNases and DNases. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 48 000, while a molecular weight of 58 000 was determined for the single band observed upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the DNase is 9.2. The enzyme hydrolyzed only DNA with a pH optimum of 8.2 and was activated by Co2+, and to a lesser extent by Mg2+ and Mn2+. Native DNA was a better substrate than heat-denatured DNA. Enzymatic digests of calf thymus and E. coli DNA yielded oligomers of chain lengths ranging from 10 to 200, with mono- and small oligonucleotides (chain length less than 5) detected only when large (100 mg) amounts of DNA were fractionated by column chromatography on diethylaminoethyl-Sephadex A-25 in 7 M urea. The digestion products contained 5'-terminal phosphate groups and mostly adenosine at the 3' and guanosine and adenosine at the 5' ends.


Structure-function relationships in TPN-dependent isocitrate dehydrogenase. I. Electron paramagnetic resonance studies of the interaction of enzyme-bound Mn(II) with substrates, cofactors, and substrate analogues. Electron paramagnetic resonance (EPR) spectra were obtained for various isocitrate dehydrogenase-Mn(II) complexes. The qualitative effects of the binding of substrates, nucleotides, and substrate analogues on the isotropic character of the electronic environment of enzyme-bound Mn(II) were subsequently investigated. The addition of isocitrate produces a markedly anisotropic spectrum whereas alpha-ketoglutarate does not alter the spectrum of enzyme-Mn(II) substantially. This suggests direct coordination of isocitrate to the Mn(II) but perphaps a different mode of binding for alpha-ketoglutarate. Other studies demonstrated mutually exclusive binding relationships between TPN and TPNH, between Mn-isocitrate and TPNH, and between HCO3-(CO2) and formate or thiocyanate. Indirect evidence supporting CO2 rather than HCO3-as the actual reactive species which binds to the enzyme in the reductive carboxylation reaction is presented on the basis of the results of the formate and thiocyanate studies. From the EPR results recorded for ternary, quaternary, and quinary enzyme-substrate complexes, correlations between the appearance of fine structure signals and the binding of individual substrates and/or nucleotides are found, and tentative assignments of such signals are made on this basis. Additional studies were conducted to determine binding constants for Mg(II) Co(II), and Co-isocitrate, and a comparison was made with kinetically determined binding constants.


Purification and properties of a 3'-phosphoryl former endodeoxyribonuclease from eggs of Asterias forbesi. A DNA endonuclease has been purified from eggs of Asterias forbesi by a simple four-step-purification procedure. The purified enzyme is at least 96% pure and is free of phosphatase, phosphodiesterase, and RNase. It has a pH optimum of 6.5 and does not require divalent cations. The enzyme produces 3'-phosphoryl and 5'-hydroxyl end groups. The products of exhaustive hydrolysis can be grouped in two fractions. The first fraction, 40%, contains a small amount of mononucleotides and di-, tri-, tetra-, penta-, and hexanucleo-tides. The second fraction, 60%, contains oligonucleotides larger than hexanucleotides.


Effect of pH on the interaction of benzoate and D-amino acid oxidase. The kinetic and equilibrium dissociation constants of the reversible binding of benzoate to hog kidney D-amino acid oxidase (DAAO) were studied at 19 degrees C over the pH range 5.3-10.5 by means of a stopped-flow apparatus and spectrophotometric titrations. A simple bimolecular reaction of the form second order-first order was observed; a two-step reaction was seen. Analysis of the pH dependence of the bimolecular rate constants and equilibrium dissociation constants is consistent with three ionizable groups which are important for benzoate binding. The pK values of the enzyme-related ionization are 6.3, 9.2, and 9.6. Analysis of the change in extinction coefficient at 360 nm indicates the pK of 9.6 can be assigned to the 3-imino group of the enzyme-bound flavin. The effect of benzoate on the apparent pK for the ionization of the 3-imino group of the enzyme-bound Fad has been reexamined. The presence of benzoate causes an apparent shift of this ionization from a pK value of 9.6 to 10.7.


Pre-steady-state kinetics of intermediate formation in the deuteroferriheme-hydrogen peroxide system. The pH dependence of formation of a peroxidatic intermediate from the reaction of deuteroferriheme with hydrogen peroxide has been determined for the region pH 8.7-10.1 from stopped-flow kinetic studies in which absorbancy changes are observed at heme monomer-dimer isosbestic points. Results are interpreted primarily in terms of the attainment of double "steady-state" concentrations of Michaelis-Menten complex I and peroxidatic intermediate I'. A linear correlation of observed first-order rate constants with alpha, the degree of dissociation of heme dimer, has been demonstrated and nonzero intercepts are obtained. Slopes and intercepts show a linear logarithmic dependence on pH which is interpreted in terms of HO2-participation both in the formation and subsequent (catalatic) decomposition of a peroxidatically active intermediate. General acid catalysis of intermediate formation is indicated from studies in phosphate, arsenate, and citrate buffer at pH 7.4-9.3. It is suggested that such catalysis may be responsible for anomalously high rates of H2O2 decomposition previously observed in phosphate buffer solution.


Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes. A combination of affinity column chromatography and preparative gel electrophoresis has been used to purify to homogeneity the two isozymes of dihydrofolate reductase from a trimethoprim-resistant strain of Escherichia coli B (RT 500). These enzyme forms are noninterconvertible and are present in crude cell lysates, but other electrophoretic species can be generated durng purification if sulfhydryl-protecting agents, such as dithiothreitol, are not present. The two isozymes, numbered form 1 and form 2 with respect to their decreasing electrophoretic mobilities, have similar molecular weights (18 500), molecular radii (21 A), and apparent Km values for reduced nico inamide adenin- dinucleotide (NADH) and NADH phosphate (NADPH). Both forms contain 2 mol of sulfhydryl/mol of enzyme which can be oxidized to intramolecular disulfide bonds. However, forms 1 and 2 differ physically in their electrophoretic mobility and isoelectric point and kinetically in their pH-activity profile, specific activity, Km for dihydrofolate, and their affinity toward a number of inhibitors.


Thermal unfolding transition of ribonuclease A measured by 2'-CMP binding. We report an approach to the problem of detecting and characterising intermediates in the unfolding of ribonuclease A. Two distinct properties of the protein are compared at equilibrium within the unfolding transition zone: (1) a physical property of the protein, the absorbance of buried tyrosine residues, and (2) a functional property, the ability to bind the specific ligand, 2'-CMP. A direct comparison of these two properties is made within the pH 5.8 transition zone, and an indirect comparison is made by using the stopped-flow instrument to sample rapidly the equilibrium properties of the pH 2.0 transition. At both pH 2.0 and pH 5.8, the results indicate that there are no intermediates in folding which have the physical properties of the native enzyme but which have lost the ability to bind a specific ligand.


Partial purification and characterization of an N2-guanine RNA methyltransferase from chicken embryos. An N2-guanine RNA methyltransferase has been purified 1000-fold from chick embryo homogenates by phosphocellulose chromatography followed by chromatography on S-adenosylhomocystein-Sepharose. The enzyme was shown to methylate the G10 position of Escherichia coli B tRNAPhe and has a Km of 3X10(-7) M for tRNAPhe and 1.38 X 10(-6) M for S-adenosylmethionine. The molecular weight was estimated to be 77 000 by gel filtration and the pH optimum was 8.0 to 8.5. Magnesium ion was not required for activity but it stimulated the rate of methylation 1.5-fold with an optimum at 12 mM. Ammonium ion stimulated activity about twofold with an optimum at about 83 mM. Sodium and potassium ions above 0.1 M were inhibitory.


Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride. Incubation of malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with ethoxyformic anhydride caused the time-dependent loss of its ability to catalyze reactions requiring the nucleotide cofactor NADP+ or NADPH, such as the oxidative decarboxylase, the NADP+ - stimualted oxalacetate decarboxylase, the pyruvate reductase, and the pyruvate-medium proton exchange activities. Similar loss of oxidative decarboxylase and pyruvate reductase activities was affected by photo-oxidation in the presence of rose bengal. The inactivation of oxidative decarboxylase activity by ethoxyformic anhydride was accompanied by the reaction of greater than or equal to 2.3 histidyl residues per enzyme site and was strongly inhibited by NADP+. Ethoxyformylation also impaired the ability of malic enzyme to bind NADP+ or NADPH. These results support the involvement of histidyl residue(s) at the nucleotide binding site of malic enzyme.


Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay. A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.


Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding. 1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.


3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. An optimized assay in homogenates of developing brain. An optimized in vitro assay of 3'-phosphoadenylysulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11, galactosylceramide sulfotransferase, formerly known as galactocerebroside sulfotransferase) activity is presented, that can be used in crude homogenate of brain tissue of various developmental stages. The enzyme activity is determined by measuring the [35S]sulfatides formed by the enzymic transfer of [35S]sulfate from 3'-phosphoadenoside 5'-phospho [35S]sulfate to galactosylceramides. The sulfatide formation at 30 degrees C is linear up to 30 min and up to a protein concentration of 1 mg per 0.5 ml assay volume. The presence of 0.4% Triton X-100 and 50 micrometer exogenous bovine cerebrosides are optimal for enzyme activity. The pH optimum of the reaction is at pH 6.5 using 0.1 M imidazole buffer. The enzyme reaction is stimulated by NaCl, KCl, MgCl2, CaCl2, MnCl2, ATP and inhibited by ADP. The developmental enzyme activity pattern of mouse brain is the same, if derived from homogenates and microsomes, respectively, under our assay conditions.


Spontaneous reactivation of acetylcholinesterase following organophosphate inhibition. I. An analysis of anomalous reactivation kinetics. The first kinetic studies on the spontaneous reactivation of Sarin-inhibited acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are reported. With increasing pH the extent of reactivation increases while the observed rate constant decreases. An analysis of the change in aging rate constant as a function of pH suggests that the aging of alkyl-alkoxy phosphonylated acetylcholinesterases is not solely acid catalyzed.


Comparative studies on immobilization of human prostatic acid phosphatase. Acid phosphatase (othophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from the human prostate was immobilized by its protein moiety on cyanogen bromide-activated Sepharose, by carbohydrate moiety on Concanavalin-A-Sepharose, and by Schiff base formation with partially oxidized carbohydrate groups on ethylenediamine-Sepharose. The highest retention of enzyme activity, 80%, was found for the noncovalent immobilization on Concanavalin-A-Sepharose. It was demonstrated that the optimal pH changes for the Concanavalin-A-Sepharose and CNBr-Sepharose-enzyme complexes are electrostratic in character. In all cases of immobilization the enzyme has higher thermostability than that for the native enzyme under the same conditions. The effects of the enzyme stabilization were interpreted in terms of the multipoint interaction between the enzyme molecule and the carrier.


Harmaline interaction with sodium-binding sites in intestinal brush border sucrase. The effect of harmaline on rabbit brush border sucrase has been studied at pH 6.8. An initial analysis in classical kinetic terms revealed harmaline to be a fully competitive inhibitor of the substrate, sucrose. In spite of this result however, the following hypothesis has been tested. Harmaline, which is positively charged in the physiological range of pH, might in fact compete, not directly with the substrate site, but rather with an allosterically-related sodium-binding site which has been postulated to be involved in the activation of sucrase by the alkali-metal ions (Mahmood and Alvarado, Arch. Biochem. Biophys. 168, 585, 1975). Because of its size, harmaline, when bound to the metal site, could at least partially overlap with the substrate site, thereby behaving as if it were an authentic fully competitive inhibitor of the substrate. This hypothesis appears to be confirmed by the fact that the alkali metals can completely reverse the inhibition caused by harmaline.


Purification of angiotensin I-converting enzyme from human lung. Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purfied using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1 mg/200 mg protein) for 2 h could be divided into three components: (i) an enzyme of molecular weight 290 000 (peak I), (ii) an enzyme of molecular weight 180 000 (peak II) and (iii) an enzyme of molecular weight 98 000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1 mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.


Isolation of buffalo muscle aldolase and comparison of its properties with those of rabbit muscle aldolase . Fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phyosphate-lyase, EC 4.1.2.13) was isolated from buffalo muscle by fractionation with ammonium sulphate and subsequent purification by phosphocellulose column chromatography using a linear salt gradient. As judged by gel filtration and electrophoresis in polyacrylamide gel, the enzyme was homogeneous with respect to size and charge. The molecular weight and Stokes radius of the enzyme were determined from its elution profile on a calibrated Sephadex column and the respective values were 162000 and 4.55 nm. The diffusion coefficient and frictional ratio were computed to be 4.8-10(7) cm2-s-1 and 1.27, respectively. The molecular weight of the polypeptide chain as measured by aodium dodecyl sulphate polyacrylamide gel electrophoresis was 40750. This taken together with the native molecular weight suggested a four-subunit model for the protein. The  N- AND C-terminal residues of polypeptide chains were identified to be proline and tyrosine, respectively. At pH 8.0 the Michaelis-Menten constant and maximum attainable velocity were found to be 8.1 muM and 27 muM Fru-1,6-P2 split/min per mg, respectively. The buffalo muscle aldolase was found to be similar to rabbit muscle aldolase in physico-chemical properties. However, the two enzymes differ significantly in pH optimum; the p optima of the buffalo and rabbit enzymes were determined under identical conditions to be 8.0 and 8.6, respectively.


Purification of methylmalonyl-CoA mutase from Propionibacterium shermanii using affinity chromatography. A novel procedure for the purification of methylmalonyl-CA mutase from Propionibacterium shermanii has been described which employs affinity chromatography on a column of immobilized vitamin B-12 linked covalently to Sepharose. The method has the advantage of being simple and rapid, thus enabling the purification of the enzyme to near homogeneity with good yields.


Enhanced heat, alkaline and tryptic stability of acetamidinated pig heart lactate dehydrogenase. Modification of 17 from 24 lysine residues in pig heart lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) with methyl aceimidate yields an enzyme derivative with enhanced stability toward meat and alkaline denaturation as well as tryptic digestion. The specific activity of the modified enzyme is only slightly reduced


Solubilization and partial characterization of UDP-N-acetylgalactosamine: globoside alpha-N-acetylgalactosaminyltransferase from dog spleen microsomes. UDP-N-acetylgalactosamine:globoside alpha-N-acetylgalactosaminyltransferase (EC 2.4.1.-) synthesizing Forssman hapten was solubilized from dog spleen microsomes by a combination of Triton X-100 treatment and sonication. The solubilized enzyme was partially purified by calcium phosphate gel, ammonium sulfate fractionation and then DEAE-cellulose column chromatography. The enzymatic activity of the purified preparation was stimulated by exogenously added phosphatidylserine, as found in the particulate enzyme. When the properties of the purified enzyme were examined in the presence of exogenous phosphatidylserine, the enzyme had an absolute requirement for Mn2+; this was not substituted by Ca2+ or Mg2+. Apparent Km values for UDP-N-acetylgalactosamine and globoside were 1-10(-5) and 5-10(-4) M, respectively. It had a pH optimum of 6.55 regardless of the presence or absence of exogenous lipids. Since the partially purified enzyme was completely free of uridine diphosphatase which was found in the particulate preparaton, the effect of UDP on the transferase activity could be studied. Thus, UDP inhibited 85% of the activity at a concentration of 1.5 mM. p-Cholormercuribenzoate inhibited over 90% of the activity at 2 mM, indicating the transferase to be SH-enzyme.


A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH. The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5.


Ganglioside biosynthesis. Characterization of CMP-N-acetylneuraminic acid : lactosylceramide sialyltransferase in Golgi apparatus from rat liver. An enzyme that transfers sialic acid from GMP-sialic acid to lactosylceramide was concentrated 40-50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents as dispersing agents. Of the numerous detergents tested, the combination Tween 80-Triton CF-54 (1 : 2, w/w) was the most effective in stimulating the reaction. Two apparent pH optima, at 6.35 and 5.5, were observed. The enzyme showed no requirement for a divalent cation. The Km values calculated for CMP-N-acetylneuraminic acid and lactosylceramide were 2.7 - 10(-3) and 1.3 - 10(-4) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane. The newly synthesized GM3, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.


Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine. The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.


Characterization and partial purification of acid lipase from human leucocytes. Hydrolysis of glycerol trioleate by human leucocytes was characterized and the enzymes responsible for this activity were obtained in a purified form by means of gel chromatography on Sephadex G-100 as well as by zonal ultracentrifugation followed by gel chromatography. The activity is localized in the granule fraction of leucocytes (15 000 X g, 20 min) and shows a sharp pH optimum at pH 5.25. As judged from the elution profile obtained by gel chromatography, two proteins are likely to contribute to the hydrolysis of glycerol trioleate. The approximate molecular weights of the two enzymes are 74 100 and 60 300, respectively. The activity is reduced in the presence of NaCl, KCl, CaCl2 as well as of p-hydroxymercuribenzoate. The enzymes are stable at -25 degrees C but loose about 50% of their activity within 48 h at 4 degrees C.


The isolation and characterization of sphingomyelinase from human placental tissue. Human placental sphingomyelinase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 000-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.


Reaction of selenium with immunoglobulin molecules. Radioactive selenite reacts with purified human and goat immunoglobulins at acidic and neutral pH. The antigenic properties of the immunoglobulins are retained during the selenium labelling as shown by immunoelectrophoresis and autoradiography. Pepsin digests of 75Se-labelled IgG possess 75Se both in the (Fab')2 fraction and in the low molecular weight peptides derived from the Fc domains. Alpha-1-acid glycoprotein, ribonuclease, and lysozyme are also labelled by this procedure. Enhancement of 75Se incorporation by urea, guanidinium chloride, mercaptoethanol, sodium sulfite and carrier selenite is interpreted as an effect of destabilization of IgG disulfide bonds. Up to 1.4 g atoms Se per mol IgG have been incorporated. We assume that selenite is cleaving disulfides by a process akin to sulfitolysis. The lability of the isolated 75Se-labelled IgG to high concentrations of mercaptans and sulfite is consistent with this idea. These 75Se-labelled proteins may be useful in structure studies and radioimmunoassay.


Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase. The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.


Circular dichroism and conformation of human alpha1-antitrypsin. The solution conformation of alpha 1-antitrypsin from human blood plasma was studied by the circular dichroism (CD) probe. The CD spectra revealed in this glycoprotein approximately 16-20% of alpha-helix, the rest of the main polypeptide chain possessing the pleated sheet (beta) and the aperiodic structures. The conformation was stable between pH 4.7 and 8.8. Reversible change in conformation was observed at pH 10.3, and more dratic denaturation occurred at pH 11.6. The environment of the side chain chromophores was strongly affected by acid at pH 2.5, whereas the main chain conformation was changed slightly. A drastic change in the CD spectra, indicating denaturation, was observed in 3.5 M guanidine hydrochloride. Sodium dodecyl sulfate was effective in disorganizing the tertiary structure and in enhancing the helix content. The phenylalanine band fine structure was observed in the native protein and also after denaturation with acid, guanidine hydrochloride and sodium dodecyl sulfate.


Iron exchange between transferrin molecules mediated by phosphate compounds and other cell metabolites. The ability of a large number of cellular metabolites to release iron from transferrin was investigated by measuring the rate at which they could mediate iron exchange between two types of transferrin. Rabbit transferrin labelled with 59Fe was incubated with human apotransferrin in the presence of the metabolites. After varying periods of incubation the human transferrin was separated from the rabbit transferrin by immunoprecipitation. GTP, 2,3-diphosphoglycerate, ATP, ADP and citrate produced the most rapid exchange of iron between the two types of transferrin, but many other compounds showed some degree of activity. Iron exchange mediated by the organic phosphates had the characteristics of a single first-order reaction and was sensitive to changes of incubation temperature and pH. The activation energy for the exchange reaction was approx. 13 kcal/mol. The rate of iron exchange from the oxalate - iron - transferrin complex was much lower than from bicarbonate - iron - transferrin. It is concluded that several organic phosphates have the capacity of releasing iron from transferrin. These compounds may represent the means by which the iron is released during the process of cellular uptake.


Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules. The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4 In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic  activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.


Purification and properties of rat brain succinic semialdehyde dehydrogenase. Succinic semialdehyde dehydrogenase from rat brain has been purified to electrophoretic homogeneity. It has a molecular weight of about 140, 000 and is composed of two apparently identical subunits. The reaction catalized by the pure protein is entirely dependent on endogenous --SH groups. The Kim (limits) for NAD and succinic semialdehyde are 2 X 10(-5) M and 1 X 10(-4) M respectively at the optimum pH of 8.6. Inhibition studies show that the reaction mechanism is a compulsory ordered on where NAD binds first followed by succinic semialdehyde.


[Role of phycobilin pigments in photosynthesis]. Experimental data on possible role of phicobilliprotein (PhBP) in photosynthesis are analysed. It is concluded that the widely spread notion about PhBP only as the light-gathering pigments which transfer the energy to chlorophyll, turned to be insufficiently substantiated experimentally until recently. At the same time the discovered ability of PhBP for reversible redox transformations together with the new data on the nature of fluorescence of blue-green algae and PhBP localization in the cell allowed a suggestion that PhBP can independently of chlorophyll or together with it directly participate in photochemical reactions of photosynthesis. A suggestion is advanced concerning possible existence of the reaction centres of PhBP in phicobillisomes.


Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168. A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg. This enzyme can not be detected in strain 168. However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.


[Genetic independence of two forms of carbonic anhydrase from bovine erythrocytes]. The two major forms of bovine erythrocyte carbonic anhydrase have been designated as CI and CII because their high activity of the C type. Separation of both forms and isolation of CI from the ethanol chloroform extract of the hemolysate were obtained by either column chromatography on DEAE-cellulose DE 23 or on DEAE-sephadex A-50. But pure preparations of the CII form were only obtained from DEAE-sephadex A-50 which separated CII from a minor component CIv1. Comparative studies of the CI and CII forms and of the minor component CIv1 strongly suggest that CIv1 is a conformational variant of CI and CII are genetic variants differing at least in their primary structure by one Arg yields Gln substitution 56 residues from the N-acetylated terminus. Based on the large variability of the proportion of the two isozymes in heterozygous individuals, the modality of the inheritance of these enzymes is discussed.


A fine-structural study of embryonic and larval development in the gymnoblastic hydroid Pennaria tiarella. 1. The pregastrulation blastomers contain electron-dense granules which become localized after gastrulation in the apices of the developing epithelio-muscle cells and persist throughout larval development. The cytoplasm of the blastomeres is organized into anucleate, membrane-delimited lobules. The lobules, which persist until six hours of development, come to contain a single, peripherally located cisterna of granular endoplasmic reticulum. Microvilli are present at the earliest stages examined and persist throughout development. Cilia are first detected at four hours. 2. Gastrulation, marked by the appearance of the mesoglea, occurs between six and eight hours of development. Basal foot processes of epithelio-muscle cells are detected by eight hours, but myonemes cannot be detected until later in development. 3. Immediately following gastrulation, mucous cells begin their differentiation from dividing cells located near the apex of the ectoderm. During their differentiation, the cells elongate toward the mesoglea. 4. By 16 hours post-fertilization, a third cell type can be detected in the ectoderm. The cell, which contains no granules, has an unusual cytoplasmic organization in which fused membranes divide the cytoplasm into parallel compartments containing a single cisterna of granular endoplasmic reticulum. 5. The findings of the present study are correlated with those of previous studies of development in Pennaria and other hydroids. The possible functional roles of the Type I granules, the cytoplasmic lobules, and the nongranular cell are discussed.


[Identification and properties of 2 forms of plant phospholipase D]. Effect of different factors (pH, temperature etc.) on the activity of phospholipase D is studied. It is found that plant phospholipase D has two forms: thermolabile (D1) and thermostable (Ds). Both forms are shown to differ in some characteristics (pH optimum, temperature optimum, calcium ions activation, activator effect, kinetic parameters, thermostavility). The contradictive literature data on phospholipase D properties are suggested to be due to the existence of two enzyme forms.


[Isolation and properties of leucine aminopeptidase from Aspergillus oryzae]. Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.


[Malic acid induction of decarboxylating NADP-malate dehydrogenase synthesis in C3-plant leaves]. The activity of decarboxylating NADP-malatedehydrogenase (E. C. 1.1.1.40) in green ethiolated pea and barley leaves and in green leaves of a pea mutant lacking photosystem II is found to be 3-fold increased after the injection of malic acid into cut plants. Protein synthesis inhibitors depressed malic acid-induced increase of the activity of "malic"-enzyme, the effect of chloramphenicol being more pronounced in ethiolated green leaves, and that of cycloheximide--in leaves of a mutant with formed photosynthetic apparatus. Possible dependence of malate-induced biosynthesis of "malic"-enzyme on the degree of NADP reduction in chloroplasts is discussed.


[Characteristics of acid ribonuclease from rat thymus chromatin]. Acid ribonuclease, free of nucleases and phosphatases, is isolated from rat thymus chromatin. The pH optimum of the enzyme is 5.0-5.5, optimal concentrations of Na+ and K+ ions are 0.05-0.15 M and 0.05 M respectively, Mg2+ inhibits the enzyme activity. The enzyme hydrolyses poly U, poly AU, cytoplasmic and nuclear RNAs, but does not attack poly A, polyG, polyC, poly A:poly U, native and denatured DNA'S. The enzyme is 3'-endonuclease, it splits the bond between the 5'-carbon atom of adenosine, guanosine and uridine and 3'-phosphate of uridilic residue. Middle length of oligonucleotides after the hydrolysis of cytoplasmic RNA comprises 10 nucleotides. Possible role of the enzyme in the processing of nuclear RNAs is discussed.


[Purification of phospholipase C from Bacillus cereus by chromatography on aminoalkylpolysaccharide adsorbents]. Purification of phospholipase C from Bac. cereus by chromatography on aminoalkylpolysaccharide adsorbents is described. The dependence of the degree of enzyme purification on the amount of ligant and effect of pH and buffer systems on the adsorption-desorption of phospholipase have been studied. At a pH below 9.0 phospholipase C is not retained by the adsorbents and is purified 4-5-fold and up to 23-fold, when aminoalkyl-Sepharose and hexamethylenediamine Sephadex are used respectively. With an increase in the pH value up to 10.0, the enzyme is bound by the adsorbent and is eluted with a 40-90% yield of activity and 7-10-fold purification. The resulting phospholipase C is highly purified and electrophoretically homogeneous. A mechanism of the enzyme-adsorbent interaction is discussed.


[pH-dependence characteristics of Ca-ATPase activity of heavy meromyosin with modified SH-groups]. Study of pH-dependence of Ca-ATPase activity of heavy meromyosin (HMM) at low and high ionic strength showed essential differences in the modifying effect of two sulfhydryl reagents, p-CMB and silver. Silver ions in conditions studied independently on pH and KCl concentration produce an inhibition of ATP hydrolysis by myosin and HMM, the shape of the pH-dependence curve remaining similar to that of the native enzyme up to 40% of blocking free sulfhydryl groups. At the same degree of binding of sulfhydryl groups with p-CMB at 0,5 M KCl the pH-dependence curve due to activation at neutral pH changes it's shape and becomes similar to that for dissociation of two ionizable groups (at neutral and alkaline regions). In contrast to this, a low or zero concentrations of KCl no activation was observed for the enzyme with 40-50% of SH-Groups modified by p-CMB and Ca-ATPase in this case seemed to be independent of pH. The data obtained suggest that SH-Groups are not included into the active site of myosin, and the activating effect observed for some sulfhydryl reagents, is due to conformational changes and it can be the result of the penetrance of the organic part of the reagent molecule into hydrophobic region of the protein.


[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components]. A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.


[Oxidation of putrescine, spermidine and spermine by diamino-oxidase from mouse liver]. Optimal conditions to determine the activity of diaminooxidase in mouse liver homogenate are described. Maximal oxidation rate for putrescine was found to take place at a concentration of 20 mM and pH 9.5, and for spermidine and spermine--at 10 mM concentration and pH 9.2. The rate of tyramine oxidation was maximal at pH 7.8. Apparent KM values were 4.98-10(-3 M, 1-10(-3) M and 0.8-10(-3) M for putrescine, spermidine and spermine respectively. Hydroxylamine did not inhibit the rate of putrescin oxidation at optimal pH value.


[Fluorescent properties of b-type ferredoxins]. Fluroescent spectra of six b-type ferredoxins of plant and animal origins were obtained. All investigated proteins do not contain tryptophan. The emission maxima of the native proteins, apoproteins prepared by various methods, and denaturated proteins are compared. The effects of pH, ionic strength and ferricyanide on the ferredoxins fluorescence were studied. "Unusual" emission at 340nm noted previously for adrenal ferredoxin was observed for spinach and Chenopodium album ferredoxins too. The localization of tyrosine fluorescent maximum at 340nm in the ferredoxins is not due to interaction of tyrosine with the iron-sulfur center. The data obtained allow to suggest that the tyrosine residues in ferredoxins have different environments.


[Relationships of photoreduction of substituted salts of 4,4'-dipyridyl by isolated chloroplasts]. Kinetic relationships of photoreduction of 4,4'-dimethyldipyridyl and 4,4'-dibenzyldipyridyl in the presence of chlorplast were studied. It was found that photoreduction leads to the establishment of photosteady state in which the rate of photoreduction is equal to the rate of dark oxidation of reduced form. Kinetics of dark oxidation of photoreduced form were investigated. The study of inhibition of photoreduction of methylviologen and potassium ferricyanide by diuron was performed. It was shown that the nature of inhibition of both reactions is completely identical. The effect of hydrogen ions concentration and gas phase composition was studied. Stabilization of low potential electron acceptors to the oxidation by molecular oxygen in the reaction of photooxidation of water is discussed.


[Some pathways of biosynthesis and degradation of polyphosphates from green algae Acetabularia mediterranea]. The activity of ATP: polyphosphate phosphotransferase was detected in free-cellular extracts of Acetabularia mediterranea. The enzyme activity in cells originally deficient in phosphorus and subsequently transferred into the phosphate-containing medium increases 5-10-fold as compared to normal. Polyphosphate degradation in A. mediterranea is probably produced by polyphosphatase, which was also detected in the free-cellular extract. It was shown that the polyphosphatase activity has two pH optima, i.e. 4.5 and 7.5, and is considerably increased when the cells are transferred into the phosphate-free medium. It is assumed that high-molecular polyphosphates involved in A. Mediterranea metabolism are responsible for regulation of orthophosphate and ATP level in the cells by ATP: polyphosphate phosphotransferase and polyphosphatase.