[Age dependence of carbohydrate tolerance and insulin secretion (author's transl)]. In 81 healthy male volunteers with normal blood pressure, normal body weight of different age (18-58 years) an intravenous glucose tolerance test with determination of blood sugar and serum insulin levels was done. Furthermore triglycerids and total cholesterol serum levels were measured. The investigations have shown: 1. With increasing age - independent from relative body weight - a highly significant decrease of glucose tolerance controlled by means of k-value according to Conard is to be measured. 2. With increasing age significant decrease of insulinsecretion after intravenous application of glucose is to be measured. Basal serum insulin levels do not significant change with increasing age. 3. With increasing age significant increase of total cholesterol serum level is to be measured, whereas triglycerid serum levels do not change. 4. Because of the significant negative correlation between k-value and total cholesterol discussion is done, that disturbances in fat metabolism are able to lead to a further deteriosation of glucoseutilisation. The investigations have shown, that the diminished glucose tolerance with increasing age is mainly related to an insufficient function of the beta-cell with increasing age.


[Manifest and latent folic acid deficiency in the aged (author's transl)]. The serum folate level of 100 aged persons (after 70) was decreased (less than 4 ng/ml) in 41% of the cases (manifest deficiency of folic acid). In the group of aged persons having normal folate values, in 41% of the cases the clearance method proved a latent folic acid deficiency, being the 15 minute value after 15 gamma/kg folic acid i.v., less than 25 ng/ml.


[Importance of space analysis of U-moment-vectors concerning ageing people and chronical heart and blood circulation diseases (author's transl)]. U-vectors are a stabil component of electric activity of the heart repolarisation. The examinations of the space sizes and direction of the U-moment-vectors give an idea about the character of repolarisation of Purkinje's fibres and papillar muscles in old age and in chronical heart and blood circulation diseases. Pathological alteration of the U-vectors appear sooner and are more explicit than the T-vectors.


[Analysis of gait in hemiplegics (author's transl)]. The gait of 31 hemiplegics is analyzed with a multiplecomponents force-plate. The healthy and paretic side differ in 5 of 7 parameters. The difference betwen the left and right side is bigger in the right hemiplegic, because in the left hemiplegic, the healthy side is more disturbed as in the right hemiplegic. That makes less differences between healthy and paretic side. The variability of the single steps is bigger in subjects with a bad gait. The method is suitable to screen the change of the gait and its progress.


[Geriatric aspects in operative treatment of intertrochanteric fractures (author's transl)]. On the basis of published data and of 702 osteosyntheses carried out using the Pohl sliding hip screw, the authors come to the conclusion that the mortality rate in geriatric patients is not correlated to the method of osteosynthesis used. Of utmost importance are immediate operation and early mobilisation. The osteosynthesis of intertrochanteric fractures in geriatric patients must facilitate early loading of the fracture site. This paper compares intramedullary nails, pertrochanteric blade plates, Y-nails and sliding hip screws, and contrasts these with the authors' own experience with the Pohl sliding hip screw.


[Morphological changes of the female urinary bladder in the elderly. Chronic infections of the urinary tract; the importance of their intensive treatment as preparatory measure for rehabilitation (author's transl)]. The urinary bladder of the elderly is showing a reduced adaptability on account of morphological changes; it is therefore more prone to infections. The morphological changes consist in a reduction of muscle fibres and in an increase of collagen fibres and in elastosis, furthermore in a sklerosis of the intramural arteries. 58% of our chronically - ill patients prove to have a chronic infection of the urinary tract.


[The pH-changes of the spinal fluid following bupivacaine (author's transl)]. The ph-changes of cerebral spinal fluid were measured following subarachnoid injection of bupivacaine 0.5% with and without adrenaline (1:200,000) during the three hours after the start of anaesthesia. At the same time drug precipitation was not detected. The ph-changes however, were pronounced but not enough to cause disturbances of the homoeostatic milieu. Especially no significant differences related to adrenaline were be found. The authors recommend bupivacaine 0.5% with and without adrenaline for spinal anaesthesia.


Experimental cryptorchidism in adult male rats: histological and hormonal sequelae. Mature male rats were rendered cryptorchid and followed for up to nine weeks during which serial blood specimens were obtained for multiple hormonal analyses; serial testicular samples were obtained as well. In contrast with control animals, cryptorchid rats showed transient rises in FSH which returned to normal at the end of the study; estrogen levels were high in the final weeks. Plasma testosterone levels were unchanged and LH levels changed little. Light and electron microscopic studies of testicular biopsy specimens showed prompt disruption of spermatogenesis which became more extensive with time. Sertoli cells underwent multiple changes including increased lipid storage and phagocytosis of spermatozoa. Comparisons are made between the sequence of hormonal changes and that of histological changes. In addition, questions are raised concerning the ways in which Sertoli cells are affected by experimental cryptorchidism.


Blood-testis barrier: maintained function of inter-Sertoli cell junctions in experimental cryptorchidism in the rat, as judged by a simple lanthanum-immersion technique. In order to elucidate the mechanisms behind the deleterious effects on the germinal epithelium of experimental cryptorchidism the hypothesis that a leaking blood-testis barrier is the cause of the damage was tested. The permeability of the specialized inter-Sertoli cell junctions to lanthanum after experimental cryptorchidism for 0.5 to 12 days was studied in the rat. In none of the time periods studied lanthanum had penetrated beyond the inter-Sertoli cell junctions. A simple lanthanum immersion technique was used. Testes of 15-days old rats (before the development of the barrier) were used as a positive control of the method, and in these testes lanthanum had penetrated up to the future lumen.


Oxygen uptake of canine whole body and hind limb with hypocapnic alkalosis. The effect of hypocapnic alkalosis induced by hyperventilation on whole-body and hind-limb oxygen uptake (VO2) was studied in dogs anesthetized with pentobarbital. In the intact dog with a self-perfused hind limb, increasing pHa from 7.41 to 7.58 increased whole-body VO2 8% and decreased hind-limb VO2 6%. Isolated hind limbs perfused with heparinized whole blood had similar decreases in VO2 with increases in arterial blood pH (pHa). However, isolated hind limbs perfused with whole blood containing citrate, phosphate, and dextrose (CPD) showed muscle twitches, had larger VO2 values at identical pHa's, and had an increase in VO2 with increase in pHa. These changes with a CPD perfusate were associated with low levels of ionized calcium (less than 0.5 mEq/1), disappeared when calcium ion spontaneously increased to 1.0 mEq/1, and could be prevented or abolished by the addition of calcium chloride, dantrolene, d-tubocurarine, or succinylcholine. These results are in accord with the findings of others regarding an increase in whole-body VO2 with hypocapnic alkalosis, but do not support a contributory role of skeletal muscle to the overall increase.


Control of gastric acidity by glycopyrrolate premedication in the parturient. The effect of premedication with the anticholinergic quaternary ammonium compound, glycopyrrolate (0.4 mg), on gastric juice pH was investigated in 23 parturients scheduled for elective cesarean section under general anesthesia, and the results were compared to a control group of 15 nonpremedicated obstetric patients and 25 parturients premedicated with atropine (0.6 mg). In the nonpremedicated control group, the mean gastric juice pH was 2.36 (SE +/- 0.23), 66% having a pH less than the critical level of 2.5. Premedication with atropine did not significantly increase the gastric pH, while in those premedicated with glycopyrrolate, the mean pH increased to 3.7 (+/- 0.35), and the incidence of pH less than the critical level dropped to 34%. The effect of glycopyrrolate on gastric juice pH was significantly increased when the premedication-induction time was prolonged to 60 to 120 minutes. It was concluded that glycopyrrolate premedication can be used in the parturient as an additional measure to safeguard against acid-aspiration syndrome.


Dirofilaria immitis and its potential mosquito vectors in central New York State. Screening of 25,822 dog blood samples indicated approximately a 1% infection rate with Dirofilaria immitis. Dipetalonema reconditum microfilariae were found in approximately 2% of 1,876 feral dogs examined. Laboratory experimentation indicated that 6 of 10 local mosquito species examined allowed successful extrinsic incubation of D immitis. Indices of experimental infection indicated Aedes triseriatus and Anopheles quadrimaculatus were excellent hosts for D immitis, but other factors considered, Aedes vexans probably served as the primary vector of D immitis. Coquillettidia perturbans and Culex pipiens-restuans group were considered unsuitable hosts and thus unlikely vectors of canine heartworm. Natural filarial infections were found in An quadrimaculatus and Ae vexans.


[Effect of tetracycline base crystallization on the conditions properties of the powders and drug forms obtained. The development of directed crystallization procedures for tetracycline base]. The study of the process of tetracycline base crystallization showed that with an increase in the rate of pH, temperature and mixed rotation changes, the specific surface of the crystalline precipitate increased with a simultaneous decrease in the bulk weight, looseness and volume density of the powder. The residual content of tetracycline in the mother solution decreased. The level of the effect of various parameters on the final results was different.


[Side effects of antibiotics in rheumatic diseases]. Complications due to antibiotic therapy in rheumatic patients subjected to season bicillin prophylaxis in Minsk within 1965--1975 and in patients with extensive collagenoses were studied. Allergic complications mainly ih the form of allergic reactions in the patients treated with bicillin-3 or picillin-5 were observed in 7.8--16.8 per cent of the patients. Within 10 years 52 cases of anaphylactic shock due to the bicillin use were recorded. Side reactions to antibiotics were observed in 16 (17.3 per cent) out of 92 patients with extensive collagenoses. For prophylaxis of the above complications it is recommended to use rational antibiotic therapy in rheumatic patients, strict registration of allergological states in the anamnesis, testing of microbial sensitivity to the antibiotics.


[Effect of the crystallization conditions of tetracycline base on the properties of the powders and drug forms obtained. The dependence of the degree of dispersion of tetracycline base on the crystallization conditions]. Characteristics of the powder dispersity of tetracycline base samples prepared by directed crystallization with variation of the process conditions were determined by the sedimentation method. It was found that the speed of the solution agitation had the maximum effect on the level and nature of the dispersity. The rate of the solution temperature and pH changing during the crystallization process had also a significant effect at low agitation speed.


Conditional killing effect of staphylococcin 1580 and repair of sublethal injury in Staphylococcus aureus. Treatment of sensitive cells with staphylococcin 1580, a bacteriocin of Staphylococcus epidermidis, rapidly induced gross changes in the permeability of the membrane. However, only a small fraction of the cells was killed when treated cells were plated on media containing low amounts of salts. Killing was greatly enhanced by increasing the amounts of small cations incorporated in the plate medium and raising the alkalinity of the medium. The effect of cations correlated inversely with the ion radius. These conditions are shown to affect the repair mechanism of the sublethally injured cells rather than the interaction of staphylococcin 1580 with the cells. A model is proposed in which the killing effect of staphylococcin 1580 is a result of the inability of cells to maintain the protonmotive force at neutral or alkaline pH as a result of the permeation of cations. Recovery of sublethal damage appears to be a complex process requiring protein and probably also ribonucleic acid synthesis and the addition of a suitable energy source.


Occurrence and distribution of bacterial indicators and pathogens in canal communities along the Texas coast. Increased construction of residential canal communities along the southern coastline of the United States has led to a concern about their impact on water quality. Pollution of such dead-end canals is potentially hazardous because of their heavy usage for recreational activities. Coliforms, fecal coliforms, and salmonellae in the surface water and bottom sediments of six selected residential coastal canals were monitored over a period of 17 months. No statistically significant relationship was observed between the organism concentrations and temperature, pH, turbidity, and suspended solids content of water. An inverse relationship between the concentration of indicator organism and salinity of water was found, however, to occur at a 99.9% level of significance. All of the microorganisms studied were found to be present in greater numbers in sediments than in the overlying water, often by a factor of several logs. Heavy rainfall resulted in large increases in the number of organisms in both water and sediment samples. Our results indicate that bottom sediments in the shallow canal systems can act as reservoirs of enteric bacteria, which may be resuspended in response to various environmental factors and recreational activities.


Development of a quantitative method for detecting enteroviruses in estuarine sediments. Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.


Production of beta-galactosidase from Streptococcus thermophilus grown in whey. beta-D-Galactosidase (EC 3.2.1.23) was extracted from Streptococcus thermophilus grown in deproteinized cheese whey. Cultural conditions optimum for maximum enzyme production were pH 7.0, 40 degrees C, and 24 h. Proteose peptone (2.0%, wt/vol) and corn steep liquor (2.8%, wt/vol) were highly stimulatory, increasing the enzyme units available in their absence from 660 U/liter of medium to 18,200 and 10,000 U/liter of medium, respectively, in their presence. There was an insignificant increase in the production of enzyme in the presence of added inorganic nitrogen and phosphorus sources. Enzymatic hydrolysis for recuction of lactose content in aqueous solution and in skim milk was studied.


Scleroderma-like changes in chronic graft vs host disease. A case of chronic graft vs host disease had scleroderma-like skin changes. Clinical progression was from poikiloderma to scleroderma, and histopathological changes and results of direct immunofluorescence were noted. It is probable that both cell-mediated (T-cell) and humoral (B-cell) mechanisms contribute to the pathogenesis of the graft vs host reaction.


Long-term esophageal function following repair of esophageal atresia. Primary repair of esophageal atresia restores gastrointestinal continuity, but does not ensure normal esophageal function. To date 22 patients, six to 32 (average 15) years after repair of their esophageal atresias, have been evaluated by personal interview and esophageal manometrics and acid reflux testing. Previous barium swallow examinations had demonstrated varying degrees of anastomotic narrowing (12 patients), abnormal esophageal motor function (11 patients), gastroesophageal reflux (two patients), and hiatal hernia (one patient). Ten patients experience intermittent dysphagia for solid foods. Seven have typical symptoms of gastroesophageal reflux. Esophageal function tests including manometry and intraesophageal pH recording, have demonstrated varying abnormalities of esophageal motility in 21 patients and moderate to severe gastroesophageal reflux in 13. Two patients have required reconstruction of the esophagogastric junction for control of severe reflux esophagitis. The unexpected high incidence of gastroesophageal reflux in these patients, coupled with their abnormal esophageal motility which impairs normal acid clearing, renders them more prone to reflux esophagitis. Careful long-term evaluation for gastroesophageal reflux and its complications is indicated following primary repair of esophageal atresia. Evaluation of esophageal function with intraesophageal pressure and pH recordings is a far more sensitive indicator of esophageal physiology than the barium swallow examination.


Interaction of drugs with apomorphine, tryptamine and norepinephrine. A new 'in vivo' approach: the ATN-test in rats. A new experimental test procedure is described for the 'in vivo' study of drug interactions with dopamine (DA), 5 hydroxy-tryptamine (5-HT) and norepinephrine (NE). The ultimate aim of this study is to provide an empirical evaluation of the relative specificity with which drugs may affect particular neurotransmitter systems, and to allow a classification of new drugs in this respect. The data indicate that, within certain dosage limits, compounds may modulate specifically a single component of the test procedure and may thus be considered as interacting specifically to modify one neurotransmitter system: i.e. DA in the apomorphine-test (e.g. pimozide), 5-HT in the tryptamine-test (e.g. pizotifen) and NE in the norepinephrine-test (e.g. phenoxy-benzamine). Higher doses of the same compounds may, however, excert effects on other neurotransmitter systems as well, and these actions may be classified as nonspecific. Thus the concept of drug specificity as applied in the present study, is not exclusive, but refers to the dissociation, in terms of doses, which exists between a drug's effect on one neurotransmitter system and its possible effects on other systems. The ATN-test separates and delineates, in terms of doses, the specific and nonspecific effects of drugs.


Cilioinhibitory effect of phenothiazines in vitro and its antagonism by Ca++. The inhibition of ciliary beating in vitro by physiologic concentrations of chlorpromazine (CPZ) and prochlorperazine (PCP) was demonstrated in two experimental systems. CPZ had a greater cilioinhibitory effect than PCP on the rotational velocity of tissue explants from the frog palate in amphibian Ringers, and on the frequency of ciliary beating in rat tracheal rings in culture medium. Addition of calcium acetate in equal molarity to CPZ, within a time limit dependent on the drug concentration, reversed this inhibition, while dibutyryl cyclic AMP did not. Simultaneous addition of Ca++ and CPZ slowed down, but did not stop, this ciliostasis, while priming with Ca++ or cyclic AMP prior to drug addition had less of an effect. These data lend support to a prevalent theory that CPZ exerts its actions by displacing calcium ions from membrane sites.


pH effects on ciliomotility and morphology of respiratory mucosa. Tracheal cilia of cows exposed, in vitro, for 20 hours to different acidities and alkalinities of sulfuric acid and sodium hydroxide, respectively, showed ciliomotility at pH values as low as 4.9, while the epithelial cells began to be expelled from the mucosa at pH 6.7, initiating a reduction of ciliary function around this pH value. In alkaline reactions, ciliostasis occurred at pH 9.76. Destroyed cilia were found above pH 10.15, while they were morphologically unchanged at pH values as low as 4.0, the lowest value examined. The first symptoms, however, of adverse effects were intracellular edema and the simultaneous occurrence of cellular polyps in both acid and alkaline reactions at pH 6.7 and 9.5, respectively. The results indicate that the early effects of air pollutants are better demonstrated by transmission electron microscopy studies than by, e.g., studies of ciliomotility.


Studies on the cell cycle of Myxobacter AL-1. II. Activities of seven enzymes during the cell cycle. The properties of seven enzymes were studied in extracts from Myxobacter AL-1. The enzymes were isocitrate dehydrogenase (E.C.1.1.1.42), succinate dehydrogenase (E.C.1.3.99.1), alkaline phosphatase (E.C.3.1.3.1), alpha-glucosidase (E.C.3.2.1.20), beta-glucosidase (E.C.3.2.1.21), beta-galactosidase (E.C.3.2.1.23), and N-acetyl-glucosaminidase (E.C. 3.2.1.30). Four of these enzymes: isocitrate dehydrogenase, alpha-glucosidase, beta-glucosidase, and beta-galactosidase are cytosolic enzymes. Succinate dehydrogenase was found to be located on the cytoplasmic membrane system, whereas alkaline phosphatase and N-acetylglucosaminidase were considered as enzymes which bind the outer membranes resp. the cell wall. During the cell cycle, all enzymes have a pattern of discontinuous activity increase. Succinate dehydrogenase and isocitrate dehydrogenase exhibit a stepwise increase of activity, whereas the other enzymes follow the pattern of a peak enzyme.


Dependence of sulphate uptake by Anacystis nidulans on energy, on osmotic shock and on sulphate stravation. Sulphate uptake by Anacystis nidulans under aerobic conditions in the light was found to be sensitive to metabolic poisons, such as N,N'-dicyclohexylcarbodiimide and carbonyl cyanide m-chlorophenyl hydrazone. It was also depressed by darkness. The sulphate absorption is an energy-dependent process. Sulphate uptake was also inhibited by chromate and selenate. Osmotic shock strongly affected sulphate uptake. This effect could be interpreted by a loss of a binding protein involved in the absorption of sulphate. Osmotic shock also depressed oxygen production in light and oxygen consumption in darkness; however, shocked cells were able to grow normally. Sulphate uptake was strongly enhanced by sulphate starvation, but this enhancement was partly prevented by chloramphenicol. Apparently sulphate starvation depressed the synthesis of a carrier involved in the transport of sulphate. During sulphate starvation the membrane potential, measured by the uptake of triphenylmethylphosphonium, increases. This may be due to changes in the membrane.


The effect of inorganic phosphate on cyanogenesis by Pseudomonas aeruginosa. The biosynthesis of hydrogen cyanide (HCN) by a strain of Pseudomonas aeruginosa is found to be significantly influenced by inorganic phosphate. Optimum HCN production occurs when the phosphate concentration is between 1 and 10 mM. Above and below this concentration the amount of HCN produced decreases sharply and at 0.1 and 100 mM phosphate low HCN production occurs. If a culture growing at 0.1 mM phosphate and producing low HCN is shifted to 10 mM phosphate, HCN biosynthesis resumes. Experiments with chloramphenicol indicate that de novo-protein synthesis is required for the process.


The role of vanadium in gree plants. II. Vanadium in green algae--two sites of action. Cells of Chlorella pyrenoidosa, derived from vanadium free agar slants, respond with great sensitivity to microamounts of vanadium, added as NH4VO3 to autotrophic liquid cultures. Between 0.01 and 1 microgram V per litre nutrient medium (2-10(-10)-2-10(-8) g-at/1), the algae respond with a continuous incrase in dry weight. At higher V-concentrations, further enhancement in biomass is accompanied by a additional increase in chlorophyll content. Maximum V-effect on both parameters was found to be at 500 microgram V/1 (10(-5) G-AT/1). Dry weight as well as chlorophyll content of Chlorella are decreased by concentrations above 25 mg V/1; 100 mg V/1 (2-10(-3) g-at/1) stop growth and cause death of the cells. The toxic threshold for the V-content in the algae was determined to be at 150-200 microgram V/g (3-4-10(-6) g-at/g) dry weight. Two different pH-optima for a positive vanadium action on dry weight and chlorophyll biosynthesis were established, the first at pH 7, the other in the range pH 7.5--8. Two sites of vanadium action in green algae are discussed.


Alpha-isopropylmalate synthase from Alcaligenes eutrophus H 16. III. Endproduct inhibition and its relief by valine and isoleucine. The alpha-isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by L-leucine and alpha-ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate alpha-ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the alpha-ketoisocaproate-mediated inhibition is discussed.


An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera. Zoogloea ramigera I-16 M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP+-linked and D(-)-beta-hydroxybutyryl CoA specific and the other was NAD+-linked and L(+)-isomer specific. The NADP+-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation for beta-hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 micrometer, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 micrometer. The incorporation of [1-14C]acetyl CoA into poly-beta-hydroxybutyrate (PHB) by bacterial crude extract (containing beta-ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of beta-ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium. These findings suggest that, in Z. ramigera I-16M, acetoacetyl CoA is directly reduced to D(-)-beta-hydroxybutyryl CoA by the NADP+-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.


Prostaglandins. A review of neurophysiology and psychiatric implications. This article reviews the function of prostaglandins (PGs) in the nervous system and discusses the possible alterations in PG metabolism as relating to mental illness. The PGs are a unique group of cyclic fatty acids whose immediate precursors are thought to function postsynaptically by inhibition or facillitation of neurotransmission through cyclase inhibition or activation, and by means of a negative feedback loop to inhibit further release of neurotransmitter from the presynaptic nerve. A review of PGs in psychiatric conditions is presented as well as a discussion of the interaction of psychoactive drugs with the PGs. The concluding section of this review discusses possible future strategies to provide insight into PG physiology as it relates to synaptic transmission in normal and pathological conditions in man.


[On the toxicology of carbromal. II. Pharmacokinetics of carbromal and its hypnotically active metabolites in the rat (author's transl)]. Oral doses up to 20 mg/kg of carbromal and of bromoethylbutyramide were rapidly absorbed in the rat. Absorption from the stomach ligated at the pyloric end was 5-8 fold less than absorption of carbromal injected directly into the small intestine. Oral doses greater than 20 mg/kg of carbromal disappeared more slowly from the gastro-intestinal tract because gastric emptying was delayed. Both carbromal and bromoethylbutyramide were able to reduce the basal tone and the acetylcholine-induced contraction of isolated rat fundus strips. Carbromal and bromoethylbutyramide distributed evenly between serum, brain and skeletal muscle. Concentrations in adipose tissue were three times those in the other three tissues. Concentrations of both carbromal and of bromoethylbutyramide in all four tissues declined at the same rate. Thus, serum concentration of either compound may be used to estimate the total body content. Intraperitoneally injected carbromal, bromoethylbutyramide and ethylbutyrylurea disappeared from the brain and from the serum with half-life of 3-4 h and 5-7 h, respectively. Traces only of unchanged carbromal, bromoethylbutyramide, or ethylbutyrylurea were excreted with urine or feces indicating rapid and extensive biotransformation of the three compounds in this species. No evidence was obtained of secretion of either carbromal or its two metabolites into the lumen of the stomach. The findings are discussed as to their relevance for acute carbromal poisoning in humans.


Separation and quantitative determination of acecarbromal, carbromal, and bromisoval as well as their main metabolites by means of high-pressure liquid chromatographic analysis. A method is described to separate and quantitatively determine bromoureides and their bromo- and non-bromo-metabolites by means of high-pressure liquid chromatography. This method allows simultaneous measurement of these substances after intake of therapeutic as well as toxic doses.


Methyl alcohol poisoning. II. Development of a model for ocular toxicity in methyl alcohol poisoning using the rhesus monkey. Rhesus monkeys were intoxicated with methyl alcohol, using an initial dose of 2 gm/kg and subsequent doses were administered in order to maintain an attenuated and prolonged state of intoxication. Arterial blood samples were drawn for methyl alcohol, formate, PO2, PCO2, and pH, which were monitored periodically throughout the course of the experiment. With the use of these procedures monkeys developed metabolic acidosis with the accumulation of formic acid in the blood and a corresponding decrease in blood bicarbonate. These animals served as models, which allowed for ocular evaluation for early signs related to methyl alcohol poisoning. A mechanism to explain toxicity is proposed and discussed.


Serotypes of pneumococci in pneumonia, meningitis and other pneumococcal infections. During a five-year period, 1965 to 1969 inclusive, pneumococci from 294 patients with acute pneumococcal infections were serotyped. Pneumococci of 33 serotypes were encountered, of which types 3 and 19 were most frequent. The spectrum of infections included pneumonia, meningitis, peritonitis, otitis media and mastoiditis, wound infection and conjuctivitis. At least 17 infections were fatal, all of which, with one exception, occurred either in infants or in adults over 50 years of age. In pneumonia, type 3 pneumococcus was predominant, being isolated from 21 of 101 patients. In 67 cases of pneumococcal meningitis, most of which were in children, the commonest type was 14. If a pneumococcal vaccine is produced for use in Australia, inclusion of serotypes 1, 3, 4, 6, 9, 14, 19 and 23 should be considered. These eight types caused 52% of the cases of pneumonia and 67% of the cases of meningitis in this study.


Potassium ion-activated hydrolysis of p-nitrophenyl phosphate in pancreatic islet-cell membranes. Hydrolysis of p-nitrophenyl phosphate was measured in a fraction enriched in plasma membranes from pancreatic islets of non-inbred ob/ob mice. Hydrolysis was stimulated by K+ (10mM) in the pH range 5--10; a small peak of K+-induced activation was observed between pH7.5 and 8. Both the K+-induced activation and the hydrolysis in the absence of K+ were Mg2+-dependent; maximum activation was obtained with 10mM-K+ plus 5 mM-Mg2+. Rb+ was as effective an activator as K+. Ouabain was inhibitory, the effect being inversely related to the K+ concentration; 0.1--0.2mM-ouabain caused about 50% inhibition in the presence of 1 mM-K+, but had no demonstrable effect in the presence of 4--5mM-K+. The K+-stimulated activity was markedly inhibited by 0.1mM-ATP, 35--140 MM-Na+, or 0.01 mM-p-chloromercuribenzenesulphonic acid. Similarities to Rb+ accumulation suggest that catalysis of univalent cation flow in pancreatic beta-cells may be coupled to a phosphoryl-transfer reaction with ATP as natural substrate or regulator.


[Determination of the KM-value of the fumarase (E.C. 4.2.1.2) with bencyclan-hydrogenfumarate as the substrate (author's transl)]. The Michaelis-Menten constant for fumarase (E.C. 4.2.1.2) has been determined by measuring the enzyme activity by the spectrophotometric method of Racker, which depends on the formation or disappearance of the double bond of fumaric acid. When using Na2-fumarate or bencyclan hydrogenfumarate (Fludilat), respectively, as a substrate, a KM-value of 1.3 X 10(-3) M was found for both substances. In a linked assay where the formation of NADH in the reaction of fumarate leads to malate leads to oxaloacetate was used as a parameter for the reaction rate, a KM-value of 1.35 X 10(-3) M was found.


Effects of clozapine and other dibenzo-epines on central dopaminergic and cholinergic systems. Structure-activity relationships. Structure-activity relationships of 16 dibenzoepines, including clozapine, loxapine, clothiapine and perlapine, have been investigated with regard to locomotor inhibition, cataleptogenesis, apomorphine antagonism, arousal inhibition, effect on striatal dopamine metabolism, and in vivo and in vitro anticholinergic potency. Thioridazine and the classical neuroleptics haloperidol and chlorpromazine were included in the study for comparison. The classical tests used to detect neuroleptic activity in laboratory animals were found to be poor predictors of possible clinical effectiveness of the dibenzo-epines.


Effect of cyclophosphamide on syngeneic transplantation of adenovirus 12-transformed tumor cells in C3H/he mice. We tested the cyclophosphamide effects against the growth of adenovirus-transformed cells and the subsequent tumor development in the syngeneic host. Cyclophosphamide did not show any effect on the tumor evolution when injected 24 and 6 hours before cell implantation. Cyclophosphamide injected 24 or 39 hours after cell implantation prevented or retarded the tumor growth. In mice bearing palpable tumors, it induced their complete regression in 85,7% of the animals, but did not effect the development of the homograft immunity.


[Diabetic ketoacidosis in children and adolescents. Clinical and therapeutical considerations in 25 severe cases]. Twenty-five serious cases of diabetic ketoacidosis, representing 23 patients, with ages ranging from 4 to 15 years are reported. School agers and adolescents were the groups most affected without existing significant predilection for sex. In 40% there was no success in finding the precipitating cause of the crisis; 32% was attributed to infectious processes, specially of the respiratory ducts and the rest, due to negligence in the application of insulin. The clinical signs showed: vomiting, dehydration, Kussamaul's respiration, sopor, stupor and in 5 cases a state of coma. Determinations of glucose, were integrated in 88% within the range of 451 to 750 mg % and the rest in lower figures. The pH in most was reported below 7.10 and CO2 lower than 10 mEq/l. Electrolytes in blood were generally evaluated within normal limits. Potassium in 20% was reported high, but we consider this was due to dehydration and because of its influence we recommend an electrocardiographic evaluation. Our classification which attempts to correlate the clinic and the laboratory is reported and our therapeutic scheme is discussed as well as the possible causes in two patients who died.


Alpha-adrenergic blocking properties of droperidol on isolated blood vessels of the dog. Concentrations of droperidol which caused a shift to the right of the dose-response curve to noradrenaline in the pulmonary artery and the saphenous vein of the dog did not affect myogenic activation by K+; they did not inhibit spontaneous activity of portal-mesenteric veins. Droperidol inhibited the contractile response to nerve stimulation, but did not affect the evoked release of 3H-noradrenaline. These experiments indicate that the vasodilator properties of smaller doses of droperidol are a result of its ability to block alpha-adrenergic receptors.


Respiratory effects of etomidate.

Respiratory effects of etomidate. The respiratory effects of etomidate 0.3 mg/kg were studied in patients premedicated with either diazepam and atropine or papaveretum and hyoscine. The incidence of apnoea was 40% in those who received the non-narcotic premedication, compared with 27% in those who received the opiate. Those premedicated with diazepam and atropine showed a significant increase in respiratory frequency which was associated with a significant increase in minute volume 4 min after induction of anaesthesia. No such increase occurred in those premedicated with papaveretum and hyoscine. It would appear that the effects of etomidate on respiration are less than those of other i.v. induction agents, but involuntary muscle movement during induction remains a problem.


Porcine malignant hyperthermia. V: Fatal hyperthermia in the Pietrain pig, associated with the infusion of alpha-adrenergic agonists. The effects of the administration of noradrenaline alone, and noradrenaline with either phentolamine or propranolol, on thermogenesis and substrate mobilization were investigated in six Pietrain (MH-susceptible) and six Large White (unsusceptible) pigs. The infusion of noradrenaline alone produced a significantly increased lipolytic response and a significantly decreased hyperglycaemic response in Pietrain pigs compared with the Large White breed. Although noradrenaline alone produced only a small increase in body temperature in both breeds, the administration of noradrenaline with propranolol in two Pietrain pigs was associated with the development of fatal hyperthermia. In a further experiment, phenylephrine or isoprenaline was infused into six Pietrain pigs. Three pigs, receiving phenylephrine, became hyperthermic and died, whereas isoprenaline had no effect on body temperature. The results demonstrate the importance of alpha-adrenergic stimulation to heat production in MH-susceptible pigs.


Urinary excretion and metabolism of pethidine and norpethidine in the newborn. In seven neonates, whose mothers were given pethidine during labour, urine was collected for the first 24-40 h of life. Urinary volume and pH, and concentrations of pethidine and norpethidine in the urine were measured. Urine flow rate was low for the first 7-22 h, and then high for about 12 h. The rate of excretion of pethidine and norepethidine was approximately parallel to the urine flow rate. However, the ratio of the rate of excretion of norpethidine to that of pethidine increased with time and the concentration of norpethidine in urine decreased first and then, after 18 h, increased significantly. These findings that the neonate can metabolize pethidine, although the rate of metabolism is probably less than in the adult. The total amounts of pethidine and norpethidine excreted in the first 24 h after birth were positively related to the dose-delivery interval in the mother for intervals up to at least 5 h. From the data it is estimated that 95% of the total pethidine transferred from the mother would be eliminated by the baby by the 2nd to 3rd day after birth.


A spectrofluorometric method for the determination of ajmaline in plasma. 1 Ajmaline was found to have maximum fluorescence at neutral pH with 300 nm excitation and 365 nm emission wavelengths (corrected). 2 The fluorescence intensity had a linear relationship to concentration up to 50 microgram ml-1 and the recovery of ajmaline after extraction from plasma was 92.5 +/- 3%. 3 Extraction of drug-free plasma and of samples containing known concentrations of ajmaline showed that drug levels in the range found clinically could be measured accurately by fluorimetry. 4 Serial plasma ajmaline concentrations were measured in a subject after intravenous injection of ajmaline (50 mg). The rates of plasma clearance of the drug were found to be similar to those obtained in previous studies.


A sensitive radioenzymatic assay for adrenaline and noradrenaline in plasma. 1 An existing radioenzymatic assay for plasma catecholamines using catechol-o-methyl transferase and [3H]-S-adenosyl-methionine has been modified resulting in a more sensitive assay for the measurement of plasma adrenaline and noradrenaline. 2 The lower limit of sensitivity for this method is 25 pg for adrenaline and 30 pg for noradrenaline/ml of plasma. 3 Resting supine (60 min) plasma adrenaline concentration was (mean +/- s.d.) 124 +/- 76 pg/ml(n=11) in males and 130 +/- 71 pg/ml (n=7) in females; plasma noradrenaline concentrations were respectively 444 +/- 129 pg/ml and 550 +/- 87 pg/ml. 4 The changes in plasma catecholamine concentrations in response to 40 degrees head-up tilt have been determined in a group of healthy normal subjects and have been shown to be related to changes in blood pressure and heart rate.


Effect of the 1,5-benzodiazepines, clobazam and triflubazam, on sleep in man. 1 The effect of the 1,5-benzodiazepines, clobazam (10 and 20 mg) and triflubazam (20 and 40 mg), on sleep was studied in six healthy males using electroencephalography for sleep measures and analogue scales for subjective assessments of well being and sleep quality. The effect of clobazam was limited to the night of ingestion. There was some evidence from subjective assessments that the effect of triflubazam may have persisted beyond the night of ingestion. 2 No effect of clobazam or triflubazam was observed on total sleep time, stage shifts in the first 6 h or latency to the first rapid eye movement period of sleep. With clobazam sleep onset latency was shortened (P less than 0.05), but this effect was not seen with triflubazam. The latency to stage 3 was shortened by both drugs. There was evidence of reduced duration of awake (stage 0) activity and drowsy (stage 1) sleep with both drugs. 3 The percentage stage 1 sleep was reduced by clobazam (10 and 20 mg) and by triflubazam (20 mg) (P less than 0.05), though the effect was not significant with triflubazam (40 mg). Clobazam (20 mg) increased the percentage stage 2 sleep (P less than 0.05), but reduced the percentage stage 3 (P less than 0.01) and stages 3 + 4 (P less than 0.05) sleep. There were no other effects on percentage of total sleep time occupied by various sleep stages or in duration (min) of sleep stages, except that the duration (min) of sleep stages, except that the duration (min) of stage 2 sleep in the second 2 h interval of sleep was increased with clobazam (20 mg) (P less than 0.01). 4 Subjects reported impaired sleep with triflubazam (40 mg) (P less than 0.05), and a sense of less wakefulness the morning after ingestion of clobazam (10 and 20 mg) (P less than 0.01) and triflubazam (40 mg) (P less than 0.05).


Clobazam, a 1,5-benzodiazepine, and car-driving ability. 1 The effects of clobazam, a new anxiolytic agent (a 1,5-benzodiazepine) on car-driving ability and other tests of psychomotor performance were investigated in a double-blind, cross-over study v. placebo in normal volunteers. 2 Clobazam (20 mg) or placebo was given nightly for six nights to ten volunteers and subjective ratings of sleep and subjective and objective assessments of behaviour and psychomotor performance on the morning following drug ingestion were recorded. 3 Clobazam significantly improved the subjective ratings of sleep induction and quality of induced sleep. 4 Clobazam did not significantly impair performance in a variety of psychomotor tests and car-driving ability. 5 The validity of the measures used and the relevance of the findings to real life car-driving situations are discussed.


Further studies of rimiterol and salbutamol administered by intermittent positive-pressure ventilation, and an important observation on the technique of using the Bennett ventilator. 1. Using the same technique of administering drugs by intermittent positive-pressure ventilation as used in previous studies a source of contamination of solutions nebulized was discovered. This was rectified by using a new ventilator and completely separate patient circuits for each solution nebulized. 2 Salbutamol 0.5% and 0.25% solutions achieved the same degree of bronchodilatation, but there was a significantly greater increase in heart rate produced by salbutamol 0.5%. 3 Rimiterol 0.5% and salbutamol 0.25% produced similar peak mean improvements in FEV and also induced the same degree of tachycardia, but the duration of these effects were significantly shorter in the case of rimiterol. 4 The sustained degree of bronchodilatation achieved by salbutamol 0.25% could not be mirrored by giving two doses of rimiterol 0.5%, the second dose 2 h after the first. 5 Rimiterol 0.5% induced a degree of tachycardia which was similar in peak effect to that observed after salbutamol 0.25%. However, in the controls the second dose of rimiterol, given 2 h after the first, was responsible for only a small increase in heart rate which was not significantly different than that after saline in the other three treatment groups.


The excretion of gamma-glutamyl transferase in pregnancy. A cross-sectional study of gamma-glutamyl transferase (gamma-GT) excretion in 316 healthy pregnant women showed the enzyme excretion (mU per mmol creatinine) was significantly higher than that of a non-pregnant control group. The increase was apparent before the end of the first trimester and continued therafter. These changes may be indicative of hypertrophy and/or hyperplasia of the nephron and be part of the overall renal adaptation to normal pregnancy. Even higher levels were recorded in three obstetric patients in whom renal complications were to be expected and this may indicate a specific type of renal damage.


Calcium-induced cooperativity of manganese binding to concanavalin A. Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.


Spontaneous, reversible protein cross-linking in the human erythrocyte membrane. Temperature and pH dependence. Changes in pH significantly affect the morphology and physical properties of red cell membranes. We have explored the molecular basis for these phenomena by characterizing the pattern of protein disulfide cross-linkages formed spontaneously in ghost exposed to acid pH or elevated temperature (37 degrees C). Protein aggregation was analyzed by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. incubation of ghosts at pH 4.0 to 5.5 (0-4 degrees C) yielded (i) complexes of spectrin and band 3, (ii) complexes of actin and band 3, (iii) band 3 complexes, i.e. dimer and trimer, and (iv) heterogeneous aggregates involving spectrin, band 3, band 4.2, and actin in varying proportions. Aggregation was maximal near the isoelectric points of the major membrane proteins, and appeared to reflect (i) the aggregation of intramembrane particles including band 3 and (ii) more intimate contact between spectrin-actin meshwork and band 3.


Kinetics of binding of oligosaccharides to a homogeneous pneumococcal antibody: dependence on antigen chain length suggests a labile intermediate complex. Temperature-jump experiments were performed with di-, tetra-, and hexasaccharides derived from type III pneumococcal polysaccharide using a homogeneous corresponding antibody IgG 45-394. A decrease in stability of the oligosaccharide-antibody complexes with decreasing chain length was observed and entirely reflected in the decrease of the association rate constants which were 1.7 X 10(4) M-1 s-1 for the di-, 3.7 X 10(5) M-1 s-1 for the tetra-, and 1.1 X 10(6) M-1 s-1 for the hexasaccharide at 23 degrees C. The dissociation rate constants for all oligomers were about 12 s-1. This marked chain-length dependence of the association rate constants as well as their low values are unexpected for a single binding step. A mechanism is proposed which consists of a fast formation of a labile oligosaccharide-antibody precomplex followed by a slow isomerization step which is induced by the oligosaccharide ligands but which is chain-length independent.


Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity. Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.


Spectrophotometric titration of a single carboxyl group at the active site of ribonuclease T1. Low-pH-induced difference spectra for ribonuclease T1, which were determined using a reference solution at pH 6, consisted of a shorter wavelength component from 270 to 285 nm that relfected an ionization having a pKa of 3.54 and a longer wavelength component above 285 nm that reflected an ionization having a pKa of 4.29. The temperature dependence of the pKa value for data at 300 nm is consistent with its representing the dissociation of a carboxyl group. In addition, the pKa determined at this wavelength significantly decreased at lower ionic strength. Similar experiments which were conducted using catalytically inactive gamma-carboxymethyl-Glu-58-ribonuclease T1 gave difference spectra having only the shorter wavelength component and were characterized by a single pKa of 3.53. It is concluded that the longer wavelength component of the difference spectra is due to the ionization of Glu-58. The pKa determined for this residue in the present study agrees with one found previously from kinetic studies which supports a role for Glu-58 in catalysis. Furthermore, the results suggest a model for the interaction of Glu-58 with histidine and tryptophan residues at the active site.


Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases. This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.


Effect of H+ on the K+ activation of adenosine-5'-monophosphate aminohydrolase. The activation of adenosine-5'-monophosphate aminohydrolase from rabbit skeletal muscle by H+ has been demonstrated. Evidence is presented which indicates that the binding of H+ and K+ is linked, in that the dissociation constant (KA) for K+ activation is reduced as the pH is lowered. Concomitantly, the pK of several enzyme functional groups is changed when K+ is added to a solution of enzyme. This change is pK results in an uptake or release of H+, depending on the pH, and shows that K+ interacts with the enzyme to achieve its effect. The uptake or release of H+ provides a simple method of following conformational changes in the enzyme following interaction of K+. The KD for K+ interaction monitored by following pH changes is the same within experimental error as that measured from kinetic data.


Phosphorylation of nuclear proteins in rat regenerating liver. In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.


Effect of pressure upon the fluorescence of various flavodoxins. The effects of hydrostatic pressure in the range of 10(-3) to 11 kbar on the fluorescence of flavodoxins from Peptostreptococcus elsdenii, Desulfovibrio vulgaris, Azotobacter vinelandii, and Clostridium MP were investigated. The first three flavoproteins showed under high pressure enhancements of flavin fluorescence of over 50 times resulting from the release of flavin mononucleotide from the protein complex. The Clostridial flavodoxin showed a very much smaller fluorescence change. At pH 7.5 the high-pressure fluorescence changes of the flavodoxins of D. vulgaris and P. elsdenii were not reversed by decompression, but in A. Vinelandii the pressure changes were over 80% reversible. At pH 5 over 80% reversibility was restored to the flavodoxins of D. vulgaris and P. elsdenii, although the pressure dependence of the fluorescence changes was very similar in the reversible and irreversible cases. The midpoint pressures in the reversible reactions were 4.7 kbar (D. vulgaris), 8.7 kbar (P. elsdenii), and 10.6 kbar (A. vinelandii) indicating specific differences in the flavin binding regions. Apparent volume changes in these reactions were 65-75 mL/mol indicating participation of a large fraction of the protein in the pressure-induced changes. The irreversible changes are not related to protein aggregation and are believed to result from a pressure-dependent covalent modification, not yet characterized, of the flavin binding region of the protein.


Occurrence of fatty acid chlorohydrins in jellyfish lipids. Fatty acid chlorohydrins are characterized as lipid components of an edible jellyfish. The four isomers 9-chloro-10-hydroxypalmitic acid, 10-chloro-9-hydroxypalmitic acid, 9-chloro-10-hydroxystearic acid, and 10-chloro-9-hydroxystearic acid were identified by gas chromatography-mass spectrometry comparison of the methyl esters and their trimethylsilyl derivatives with known synthetic samples. Two additional isomers, 11-chloro-12-hydroxystearic acid and 12-chloro-11-hydroxystearic acid, were also found in the lipid by the identification of the expected mass spectral fragments of the trimethylsilyl (Me3Si) derivative of their methyl esters. These six isomeric compounds represented approximately 1.4% of the total extractable jellyfish lipid and were released from the lipid as methyl esters by boron trifluoride-methanol treatment. These isomers account for only about 30% of the organic chlorine in the lipid. Evidence is given that the remaining organic chlorine is also present as fatty acid chlorohydrins containing more than one hydroxyl group.


Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. I. General kinetic analysis. Steady-state kinetics of electron transfer through the cytochrome chain of uncoupled ultrasonic submitochondrial particles at different pH values has been studied. Rate constants calculated from the Pring equation (ki' = V/PirPi+1ox) increased with the increase of the rate of the process. As in the previous work (Saks, V. A., Kupriyanov, V. V. and Luzikov, V. N. (1972) Biochim. Biophys. Acta 283, 42-53) this dependence was linear, but only at comparatively low rates of electron transfer. To explain the experimental data several kinetic models, based on the assumption that respiratory chains are activated when functioning, have been proposed and analysed. The best agreement with the experimental data was obtained for the model suggesting that the rate of activation of the carriers is directly proportional to the overall rate of electron transfer and to the proportion of non-activated respiratory chains in the system. Hence it appeared that electron transfer through already activated chains entailed activation of adjacent non-activated chains. This model allowed rate constants for non-activated (ki) and activated (ki) states of the carriers, as well as the life-time of the activated carriers (tau) to be determined.


Conformation-dependent participation of the protein in electron equivalent transfer to cytochrome c. When ferricytochrome c is reduced by H atoms (produced by pulse radiolysis) at neutral pH where it is in a closed protein configuration, a considerable percentage of the reduction proceeds through electron equivalent transfer via the protein. At pH 2.0, where cytochrome c is in an open configuration, H atoms reduce by adding directly to the heme porphyrin. The intermediate then observed is identified through similarity with that formed on ferriheme alone.


Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors. Using artificial electron donors and acceptors, it is shown here that the major HCO3- effect in the Hill reaction is after the "primary" electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acdeptor Q- to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q-, show no significant bicarbonate stimulation of electron flow. However, a 6-7 fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3- (3',4'-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.


Interactions of Tetrahymena dynein with microtubule protein. Tubulin-induced stimulation of dynein ATPase activity. The ATPase (EC 3.6.1.3) activity of 30 S dynein from Tetrahymena cilia was remarkably stimulated by porcine brain tubulin at pH 10. The activity increased with increasing concentration of tubulin until the molar ratio of tubulin dimer to 30 S dynein reached approx. 10. The optimum of the ATPase activity of 30 S dynein in the presence of tubulin was 1-2 mM for MgCl2 and 2 mM for CaCl2. Increasing ionic strength gradually inhibited the stimulation effects of tubulin. Activation energies of 30 S dynein in the presence and absence of tubulin were almost the same. At the temperatures beyond 25 degrees C stimulation effects of tubulin disappeared. ATP was a specific substrate even in the presence of tubulin. In kinetic investigations parallel reciprocal plots were observed in a constant ratio of divalent cations to ATP of 2, indicating that tubulin was less tightly bound to 30 S dynein in the presence of ATP than the absence. The similar results were obtained at pH 8.2. 14 S dynein and the 12 S fragment which have poor ability to recombine with outer fibers were also activated with brain tubulin.


Non-covalent cross-linking of lipid bilayers by myelin basic protein: a possible role in myelin formation. Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, L-alpha-dimyristoyl phosphatidylcholine and DL-alpha-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15-18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associated species. A similar interaction was observed with diacyl phosphatidylserine vesicles. These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myelin, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.


Contrast manifestation of alkaline phosphatase and 5'-nucleotidase in plasma membranes isolated from rat liver and ascites hepatoma. 1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.


Partition of catalase and its peroxidase activities in human red cell membrane: effect of ATP depletion. Partititon of catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase EC 1.11.1.6) and peroxidase (donor:hydrogen-peroxide oxidoreductase EC 1.11.1.7) activities between the red cell membrane and the cytosol were studied under various experimental conditions. A small but significant amount of catalase (1.6%) was retained on human red cell membranes prepared by hemolysing washed red cells with 30 volumes of 10 mM Tris buffer, pH 7.4. Membrane -bound catalase had a relatively higher peroxidase activity than the soluble enzyme fraction. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the solubilized membranes demonstrated catalase to be a single band with a molecular weight of 60 000. Membranes prepared from adenosine triphosphate-depleted red cells depicted a two to three-fold increase in catalase activity, as well as an increase in 60 000 molecular weight band on polyacrylamide gel electrophoresis. The extra amount of retained catalase was a less efficient peroxidase than found in fresh membranes. The binding of catalase to ATP-depleted red cell membranes was dependent upon both pH and hemolysing ratio. Red cells incubated at pH 7.1 demonstrated a decrease in bound catalase, as did membranes prepared from red cells hemolysed at 1:100 dilution. beta-Mercaptoethanol decreased the catalase activity in the membranes and increased the odianisidine peroxidase activity without any significant effect on the 60 000-dalton band.


Polyribonucleotides containing thiopurines. Synthesis and properties of poly(1-methyl-6-thioguanylic acid). The synthesis of 1-methyl-6-thioguanosine 5'-diphosphate and its conversion to poly(1-methyl-6-thioguanylic acid) by means of polynucleotide phosphorylase are described. The polymer exhibited cooperative behavior (Tm = 294 K in the absence of added NaCl) characteristic of a highly stacked single-stranded helical array. In a high salt environment (0.5 M NaCl) the melting was much less cooperative and gave a higher Tm (313 K); this is suggestive of interstrand aggregation involving hydrogen bonding. The polynucleotide exhibited a remarkably high pKa (6.2) compared to that of the mononucleotide (2.6), and formed a very stable acid structure (Tm = 356 K in 50% ethylene glycol). Comparisons with poly(1-methyl-6-thioinosinic acid) and poly(6-thioguanylic acid) establish that both the 2-amino group and the 1-methyl group are required for the formation of the stable acid structure.


Biphasic effects of translational inhibitors on liver tyrosine aminotransferase. Low doses of cycloheximide or emetine cause rat liver tyrosine aminotransferase activity to rise up to twice the control levels in 2 h. By contrast, in the same interval no changes, or only a slight decrease, are produced by either drug at high dosage. Adrenalectomised animals display the same pattern of response. High doses of either antibiotic virtually afford a complete inhibition of 14C-labelled amino acid incorporation into liver and plasma proteins, whereas no more than a 30% decrease is observed with low doses. When administered in the course of the induction by cortisol, high doses of inhibitor prevent any further change in tyrosine aminotransferase activity, stabilising it at the level already attained; low doses, while slightly affecting the synthetic phase evoked by cortisol, drastically interfere with the deinduction. Six hours after various doses of either inhibitor the tyrosine aminotransferase activity is markedly increased, this late effect being largely dependent on the presence of adrenals. The amino acid incorporating actitivy of the liver may exceed that of controls, as observed particularly after small doses of emetine.


Fluorimetric assay of tobacco leaf dehydrogenases with resazurin. A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.


Purification and characterization of 2-oxoaldehyde dehydrogenase from rat liver. The partial purification (123-fold) of 2-oxoaldehyde dehydrogenase (2-oxoaldehyde:NAD(P)+ oxidoreductase, 1.2.1.23) from rat liver was carried out using a purification procedure which involved (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, Blue-Dextran affinity chromatography and CM-Sephadex chromatography. A single form of the enzyme was observed, mol. wt. approx. 50000 by gel chromatography. 2-Oxoaldehyde dehydrogenase appears to be highly specific for NADP+ and methylglyoxal. No activity is observed in the absence of certain amines which have vicinal amino and hydroxyl groups. The only known amine which activates the enzyme at physiological pH is L-serine methyl ester, suggesting that the regulation of this enzyme in vivo may require a derivative of serine.


Oxidation of hypoxanthines, bearing 8-aryl or 8-pyridyl substituents, by bovine milk xanthine oxidase. 1. Hypoxanthines, bearing at position 8 aryl or pyridyl substituents, are converted by bovine milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) into the corresponding xanthines at low rates. Oxidation is accelerated considerably when the 8-pyridyl substituents are quaternised. 2. In the enzymic oxidation of quaternary 8-pyridylhypoxanthines a lag phase precedes the attainment of a constant, maximal reaction rate. It is assumed that the delay is due to a relatively slow conformational change in the active enzymic center. 3. In 8-(3'-N-methylpyridinio)xanthine betaine, also the pyridinium moiety is attacked at high pH (9-11) to yield an N-methyl-2-pyridone. The analogous pyridone is the only oxidation product of 1-methyl-8-(3'-N-methylpyridinio)-hypoxanthine betaine, which is not attacked in the pyrimidine ring. 4. The cationic substrates are attracted to the enzyme by an anionic group, which probably forms an ion pair with a protonated amino group in or near the active center.


Evidence about the catecholoxidase activity of the enzyme ascorbate oxidase extracted from Cucurbita pepo medullosa. Pure ascorbate oxidase (L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) isolated from Cucurbita pepo medullosa, which is known to be specific for ascorbic acid, shows a secondary catecholoxidase activity at approx. pH 6.7. This activity was tested against natural and synthetic compounds possessing a catechol-like structure. Among natural compounds (+)-catechin furnishes the same complex oxidation mixture obtained with other oxidases. Among synthetic compounds, 3,5-di-t-butylcatechol and 4-t-butylcatechol give the corresponding o-quinones. The significance of this secondary activity in the darkening process of fruits and vegetables which contain ascorbate oxidase is also discussed.


Canine thyroid fucokinase.

Canine thyroid fucokinase. A radiometric assay was developed for fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) based on the conversion of L-[14C]fucose to L-[14C]fucose 1-phosphate which is trapped and counted on ion exchange paper. This assay was used to detect the presence of a fucokinase in canine thyroid tissue which was subsequently purified 2754-fold over the crude tissue extracts. The product of the fucokinase was identified as the beta-anomer. The pH versus activity curve for the enzyme appears biphasic with optima at pH 6.5 and pH 8.25. The enzyme was shown to be highly specific for L-fucose with a Km of 2.6 - 10(-5) M at pH 8.25. It was shown to be absolutely specific for ATP as a phosphate donor with a Km of 6.3 - 10(-4) M at pH 8.25. The enzyme requires a divalent cation. Mg2+ is slightly more effective than Mn2+ in meeting this need. The molecular weight of the enzyme has been determined to be 494 000 +/- 12 400.


The mode of binding of potential transition-state analogs to acetylcholinesterase. Phenylacetone, 4-phenyl-2-butanone, and 4-oxopentyltrimethylammonium chloride were tested as potential transition state analogs for eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). Phenylacetone is a competitive inhibitor of the enzyme but not a transition state analog, since its binding constant is similar to that for the substrate phenyl acetate. 4-Phenyl-2-butanone binds 6-18 times more tightly than the inhibitors 4-phenyl-2-butanol and N-benzylacetamide and the substrate benzyl acetate and also blocks inactivation of the enzyme with methanesulfonyl fluoride. However, its binding is independent of pH in the range 5-7.5, whereas both V and V/Km for benzyl acetate hydrolysis decrease with decreasing pH in this range. These data indicate a specific but weak interaction between the ketone carbonyl and the enzyme, but probably do not justify considering this compound a transition state analog. 4-oxopentyltrimethylammonium iodide has previously been shown to bind about 125 times more strongly than the substrate acetylcholamine. It also binds about 375 times more strongly than the alcohol 4-hydroxypentyltrimethylammonium iodide. Furthermore, the ketone protects the enzyme from inactivation by methansulfony fluoride, while the corresponding quaternary ammonium alcohol accelerates this inactivation reaction. This additional information confirms that the ketone is a transition state analog.


pH dependence and solvent isotope effects in the hydrolysis of phosphomonoesters by human prostatic acid phosphatase. The pH dependence of the human prostatic acid phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate and beta-glyceryl phosphate has been studied over a wide range of pH and the values of Km and V calculated with the aid of the Cleland HYPER program. The pH dependence of Km shows the effect of substrate ionization: pK values of 5.6 and 6.4 are observed as for the respective values of free substrates. The pH dependence of both Km and V for each substrate reveals the involvement of an ionizable group in the ES complex which is ascribed to a phosphohistidine-enzyme intermediate. The small deuterium solvent isotope effects which are observed on V are consistent with values observed for solvolysis of phosphoramidates. The measured data for Km indicates limits on burst-titration experiments of prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2).


Proton magnetic resonance studies of histidines in human, rhesus monkey, and bovine carbonic anhydrases. Histidine C-2 proton resonances in rhesus monkey carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) and bovine carbonic anhydrase were investigated using 270-MHz proton magnetic resonance. The results suggest that there are extensive three-dimensional homologies between the human B and rhesus B enzymes and between the human C and bovine enzymes. Resonances from solvent exchangeable protons have been observed in the 11-16 ppm range in the NMR spectra of human carbonic anhydrases B and C and bovine carbonic anhydrase. Up to five of these are sensitive to changes of pH and the presence of inhibitors. Three of these resonances are assigned to NH protons of the metal coordinated imidazole groups. These results are discussed in relation to various models for the catalytic mechanism of carbonic anhydrase.


Kinetic analysis of calcium activation of brain acetylcholinesterase forms. Calcium activation of acetylcholine hydrolysis by bovine brain acetylcholinesterase (Acetylcholine hydrolase, EC 3.1.1.7) forms has been analyzed in terms of changes in kinetic constants and thermodynamic activation parameters. De-acetylation was determined to be the major rate-influencing step in acetylcholine hydrolysis by both 60 000- and 240 000-dalton forms of the brain enzyme and 10 mM Ca2+ increased the rate constant for this step (k+3) by approximately 30% for both forms. For the smaller acetylcholinesterase form the effects of Ca2+ on de-acetylation was equivalent to its effect on the overall rate constant (k) and occurred without an effect on pK. In the case of the 240 000-dalton species, the overall rate constant was increased by Ca2+ by 33% at pH 8.0 and 81% at pH 7.25 and involved a pK shift of -0.2 pH units. For both enzyme forms the rate constants for acetylation (k+2) were increased by Ca2+. Thermodynamic analysis suggested that Ca2+ activation of the acetylation step was entropically driven. Differences between the two enzymes forms in terms of Ca2+ appear to result from association of low molecular weight species.


Resolution of independently titrating spectral components in the ultraviolet circular dichroism of subtilisin enzymes by matrix rank analysis. The ultraviolet circular dichroism of di-isopropylphophoryl-subtilisins Carlsberg and Novo (EC 3.4.21.14) has been examined as a function of pH. The CD of these enzymes below 260 nm is invariant over the pH interval 4 to 12, below or above which spectral changes occur suggesting a transition to a random coil form. Above pH 8 contributions due to the ionization of tyrosyl residues appear in the CD above 260 nm as bands shifted to longer wavelengths. Three independently titratable components, obtained by matrix rank analysis, account for the observed CD spectral changes above 260 nm of Dip-subtilisin Carlsberg in the pH interval 8 to 12. By contrast, two components were derived for the Novo enzyme. The identities of the matrix rank components were surmised from their apparent pKa values. One component of both subtilisin enzymes corresponds to the CD of the "buried" or irreversibly titratable tyrosyl residues of the enzyme. The other matrix rank components correspond to the CD of the "exposed" or freely ionizable tyrosyl residues. These residues are optically active only in the ionized state. Two types of "exposed" tyrosyl residues, arising because of differing sensitivity to the ionization of the "partially buried" or abnormally titrating tyrosyl residues, are evident in Dip-subtilisin Carlsberg. A pH-induced local conformational change in this enzyme is proposed to account for this behavior. The "partially buried" tyrosyl residues of both subtilisins appear to be devoid of optical activity in either the tyrosyl or tyrosylate form.


Some characteristics of soluble fatty acid synthesis in germinating pea seeds. Soluble fractions from germinating pea synthesize palmitic acid de novo and stearic acid by elongation. Malonyl CoA, acyl carrier protein and NADPH are required for both reactions. In contrast to some other plant systems, no requirement was found for divalent cations. On the other hand, the formation of both stearate and palmitate was inhibited by sulphydryl reagents and palmitate elongation was sensitive to arsenite. The products of the reactions were examined and found to be principally acyl-acyl carrier proteins and unesterified fatty acids. Unlike the pea microsomal fractions, the soluble enzymes are stimulated only slightly by the addition of exogenous lipids. The substrate for palmitate elongation is palmitoylacyl carrier protein, which is quantitatively elongated to stearate. Comparisons are made with membrane-localised fatty acid synthesis from the same tissue.


Immunoelectrophoretic studies on pig intestinal brush border proteins. Brush borders were prepared from pig intestinal mucosa and the membrane proteins solubilized with either Triton X-100 or papain. Proteins, thus released, were used as antigens to raise antisera in rabbits. The immunoglobulin G fractions were isolated and shown by the double layer immunofluorescence staining technique to react only with the brush border region of the enterocyte. The antibodies obtained were used in immunoelectrophoretic studies on the brush border proteins. Eight hydrolytic activities were identified by the use of histo-chemical staining methods. These were the microsomal aminopeptidase (EC 3.4.11.2), aspartate aminopeptidase (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.X), lactase (EC 3.2.1.23), glucoamylase (EC 3.2.1.3), sucrase (EC 3.2.1.48), isomaltase (EC 3.2.1.10) and alkaline phosphatase (EC 3.1.3.1). In addition, at least four faint immunoprecipitates were formed but none of these were identified.


Properties of trout hemoglobin covalently bound to a solid matrix. This paper reports the ligand binding properties of the major hemoglobin component from trout (Salmo irideus) covalently bound to a solid matrix (Sepharose or Sephadex). A comparison between the functional properties of this protein in solution and of the protein-matrix complex shows significant changes although the basic properties of the molecule are maintained on covalent binding to Sepharose (or Sephadex). Thus the Root effect, characteristic of Hb trout IV, is still present while the heme-heme interactions are, on the average, smaller in the matrix bound protein as compared to the soluble form. No differences in the O2 binding properties were observed when the protein was coupled to the resin, as the ligand bound or as the ligand free derivative. Although an unequivocal interpretation of the data is made difficult by the lack of information on the number and identity of the groups involved in the coupling, the main changes in the protein functional properties may be related to the chemical modifications "per se" more than to the immobilization imposed to the macromolecule by coupling to the matrix. Structural changes which mainly involve perturbation of the tertiary structure of the molecule may qualitatively rationalize the data.


Resonance Raman study of the pH-dependent and detergent-induced structural alterations in the heme moiety of Rhodospirillum rubrum cytochrome c'. The resonance Raman spectra and the structures of the heme moiety of Rhodospirillum rubrum cytochrome c' were investigated for its five states characterized by absorption spectra; Types-a and -n of the reduced form and Types-I, -II, and -III of the oxidized form. The frequency of the ligand-sensitive Raman line suggested the coordination of lysine (Nepsilon) at the sixth position of the heme iron of Type-n. The sixth ligand of Type-III was deduced to be either lysine or histidine but would not be methionine. Type-a and Type-II gave the Raman spectra of rather normal high spin type but Type-I was unusual in the sense that the frequencies of the Raman lines associated primarily with methine-bridge CC-stretching vibrations were relatively high in comparison with those of other high spin hemoproteins. Type-I was converted directly to Type-III upon the addition of SDS or 2-propanol but the conversion occurred via Type-II when pH was increased. Structural difference between the high spin hemes of Type-I and Type-II was discussed in detail.


Hydrogen ion titration and amino acid analysis of hemocyanin from the spiny lobster Jasus edwardsii. Potentiometric and spectrophotometric titrations, isoelectric focusing and amino acid analyses, have been made on the hemocyanin from Jasus edwardsii. Counts of acidic neutral and alkaline groups were made from the titrations, enabling comparisons to be made with the amino acid analysis. Thermodynamic analysis of the data indicated that changes in the native protein structure took place at pH 4.0, 8.4 and 10.7. These observations are discussed in terms of dissociation, shifts in pK and conformational changes in the protein.


Effect of tyrosine ionization upon biological activities of angiotensin II and two new peptide analogues. The role of the tyrosine side-chain in the smooth muscle contracting activity of angiotensin III was investigated by determining intrinsic activities and ED50 values of [4-(3-chlorotyrosine)]angiotensin II and [4-(3-benzyltyrosine)]angiotensin II in the isolated guinea-pig ileum and rat uterus. [4-(3-chlorotyrosine)]angiotensin II activity was compared with that of angiotensin II at different pH values, in which the ratio of their degrees of phenolic ionization varied. The results indicated that deprotonation of the phenolic group hinders binding to smooth muscle cell receptors, but not triggering of the response by the hormone-receptor complex. Steric hindrance by the benzyl substituent in [4-(3-benzyltyrosine)]angiotensin II reduced both receptor-binding and triggering of the response.


Increased oxygen affinity for hemoglobin Sawara: alphaA4(6) aspartic acid replaced by alanine. The oxygen binding property of Hb Sawara (alphaA4 Asp replaced by Ala) was studied at different pH values with and without addition of 2,3-diphosphoglycerate. The oxygen affinity of Hb Sawara was shown to be increased, the difference of the log P50 value between normal and abnormal hemoglobins being 0.37 at pH 7.0. Both the magnitude of the alkaline Bohr effect and the effect of 2,3-diphosphoglycerate upon oxygen affinity of Hb Sawara were comparable to those of Hb A. The amino acid substitution of alanine for alphaA4 aspartic acid might result in the loss of a stabilizing force for ionic interaction between the alpha-amino group of NA (1)alpha1 valine and the alpha-carboxyl of HC3(141)alpha2 arginine in the deoxy-form.


Thermodynamic study of the formation of adenine nucleotide-manganese complexes. I. "pH stat" titration method results. The thermodynamics associated with the Mn2- -ATP, Mn-ADP- and Mn-AMP complex formation reactions determined from K potentiometric measurements at I = 0.2 are reported for the temperature range 1--45 degrees C. The K values increase with the length of the phosphate chain and with temperature. The limits and the best conditions for use of the "pH stat" titration method are discussed. Comparison with the results obtained by potentiometric and calorimetric methods in the case of Mg-nucleotide complexes is made.


Thermodynamic study of the formation of adenine nucleotide-manganese complexes. II. Calorimetric results. Enthalpic variations in the formation of adenine nucleotide-manganese complexes, as measured by microcalorimetry, are reported. All the results are obtained in the temperature range 6--30 degrees C at I =0.2 and pH values 7.00 or 7.50. All the reactions are endothermic and the deltaH values increase with the length of the phosphate chain and with temperature. The deltaH values are compared with those previously obtained for adenine nucleotide-manganesium complexes. The comparison between calorimetric and potentiometric deltaH values is made. The divergence observed between these results at low temperature leads us to assume the formation of nucleotide aggregates induced by the presence of manganese ions. This hypothesis is confirmed by differential ultraviolet spectra.


[Isolation and characterization of beta-D-galactosidase from gastric juice]. A method for beta-D-galactosidase isolation from cattle gastric juice has been developed. Gastric juice mucus was removed by and addition of equimolar amounts of Na2HPO4 and CaCl2. The removal of proteases and other proteins was achieved by the treatment with resins KB-51X2 and AN-22. The resulting preparation had specific activity of 0.14 units per mg of protein. Gel filtration on Sepharose 4B led to an increase of specific activity of the preparation up to 0,4 units per mg of protein. Some properties of the beta-D-galactosidase obtained were compared to relation of lactose and o-nitrophenyl-beta-D-galactoside (pH optima, temperature optimym and thermostability).


[Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase]. A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.


[Purification and some properties of isozymes 1 and 5 of lactate dehydrogenase from fox heart and skeletal muscles]. An improved method  is described for the isolation of isozymes 1 and 5 of lactate dehydrogenase (LDH) from heart and skeletal muscles of foxes. The method includes salt fractionation with ammonium sulphate, chromatography on DEAE- and CM-celluloses and affinity chromatography on AMP-Sepharose. The preparations of LDH isozymes 1 and 5 turned out to be homogeneous both in 7,5% polyacrylamide gel electrophoresis and under immunodiffusion analysis. It is shown that the pH optimum for LDH-1 is 10.2-10.4 for LDH-5 it is 9.5-9.6 in the case of the direct reaction, and the pH optimum is 7.6-7.8 for LDH-1 and 7.3-7.4 for LDH-5 in the case of reverse reaction. The values of Mikhaelis constants were determined for substrates and coenzymes in direct and reverse reactions. It is found that the excess of lactate and pyruvate causes substrate inhibition of both LDH-1 and LDH-5. The activities of LDH-1 and LDH-5 showed an unexpected similar sensitivity to the inhibitory effect of high pyruvate concentrations.


[Photogeneration of a 2-vector transmembrane proton gradient in Halobacterium halobium R1 cells]. Analysing 4 phases of light-dependent change of pH in cell suspension of H. halobium R1 it has been found that an increase of pH I when light is switched on and a decrease of pH II during further illumination are proportional to the light effect and are energy-dependent. Neutralization of these changes (phases III and IV) proceeds spontaneously in the darkness. These data show that the transmembrane gradient of protons is generated in two directions--delta pH and +delta pH, simultaneous presence of which points to the local character of one of them. Generation of -delta pH associated with light energy utilization by the cells is considered as a result of neutralization of the negative charge and +delta pH1 (latent), appearing due to a change in bacteriorodopsin conformation under illumination. It may be also suggested that the yield of protons from the cells of pHII and generation of +delta pH result from the discoupling of photoprocesses and energy utilization by the cells, containing bacteriorodopsin.


[Comparative study of NADP-reductase properties in two species of purple bacteria]. Unlike Rhodospirillum rubrum, the highly purified preparations of NADP-reductase Thiocapsa roseopersicina are capable of reduction of cytochrome c though they do not catalyse diaphorase reaction in the presence of methyl viologen or benzyl viologen and NADH. T. roseopersicina reductase has more high temperature optimum (50-65 degrees) and more high thermal stability (65 degrees) and it is capable to catalyse diaphorase and menadione-reductase reactions under more high pH values (11.0-12.0) than NADP-reductase of R. rubrum. NADP-reductase of T. roseopersicina is more stable under storing than the enzyme from R. rubrum: the semi-inactivation period of the enzyme when storing in Ar or the air is about 10 and 4 days, respectively, and it takes about three days for R. rubrum.


[Functional role of parvalbumins in regulating Ca2+ in the contraction--relaxation cycle of vertebrate skeletal muscles]. On the basis of parvalbumin property to change Ca2+-binding constant within the pH-region from approxim-tely 7 to approximately 8 a hypothesis that parvalbumins are pH-regulated Ca2+-depot in muscles has been stated. The addition of parvalbumin to native actomysin complex at pH approximately 7 in the presence of ATP and Ca2+ results in partial inhibition of its superprecipitation. The change of pH up to approximately 8 leads to the restoration of actomyosin superprecipitation.


[Virioimmunoassay for studying the interaction of diphtheria toxin with cell membranes]. A model for studying the interaction of diphtheria toxin with cell membranes includes immobilization of purified cell membranes on Sephadex G-25, adsorbtion of toxin on the membranes in the presence of protective colloid, and subsequent detection of adsorbed toxin by means of virioimmunoassay. Diphtheria toxin adsorbed rapidly on membranes of both sensitive (HeLa, macrophages) and resistent to tis action cells, but not on stromaof human erythrocytes. The rate of interaction depends on the concentration of toxin and the temperature of incubation. Adsorbed toxin may be eluted by acidic buffer, 8 M area and 4 M guanidin. HCl, but not by triton X-100, tween 20 and 80, sodium dodecylaulfate and basic buffer.


[Precipitation of neutral alpha-glucans and separation of mixtures by dimethyldodecylbenzylammonium chloride]. The solubilization of precipitated complex of alpha-polyglucane-dimethyldodecylbenzylammonium chloride in excess of a solution of the salt was found to be dependent on pH. The complexes of glycogen having a more branching structure are dissolved much more readily than the amylopectin complexes. The conditions for separation of mixtures of these two alpha-glucanes were found. The purity of glycogen and amylopectin obtained after separation from the mixture was established both by comparing the absorption spectra of their iodinepolysaccharide complexes and by separation of radioactive amylopectin and non-radioactive glycogen mixture.


[Regulation of Na, K-ATPase activity by monovalent cations]. A deviation from optimal conditions of the Na, K-ATPase reaction results in a drastic change in the plot: enzyme activity versus Na/K ratio. Acidification of the medium and a decrease in Mg2+ concentration and temperature results in two peaks on the curve at Na/K ratio of about 1 and at Na/K ratio greater than 4. The enhancement of pH of the medium and increase in Mg2+ concentration decreases the first peak and increases the second one. A comparison of these curves for hydrolysis of ATP, UTP and p-nitrophenylphosphate and temperature dependence of the hydrolysis of the substrates suggest that the anomalies observed may be accounted for the Na+ effect on the K-sites or K+ effect on the Na-sites under conditions when cation-binding sites are heterogeneous.


[Reversible dissociation of malate dehydrogenase from plants]. It was demonstrated that 0.2 M citric acid (pH 2.5) inactivates highly-purified malate dehydrogenase from tea leaves; the degree of inactivation depends on temperature and time of incubation. The enzyme activity is restored by certain inorganic salts, the degree of reactivation being dependent on pH, ionic strengths of salts and duration of enzyme incubation with both inactivating and reactivating agents. Urea and guanidine hydrochloride also have a reversibly inactivating effect on the enzyme. The degree of inactivation depends on their concentration and incubation time. In the latter case reactivation of enzyme is achieved by dialysis or 20-40-fold dilution of the enzyme preparation. A kinetic study demonstrated that inactivation of enzyme by the above-mentioned agents is due to the enzyme dissociation into 4 catalytically inactive subunits with molecular weights of 17 500 +/- 1000, which under certain conditions are capable of reassociating into an active molecule of enzyme with completely restored native conformation.