Species distribution and properties of hepatic phenylalanine (histidine):pyruvate aminotransferase. Hepatic phenylalanine(histidine):pyruvate aminotransferase activity is much higher in the mouse and rat than in other animal species (human, guinea-pig, rabbit, pig, dog and chicken). The activity is elevated in the mouse and rat by the injection of glucagon but not in other species (guinea-pig, rabbit and chicken). The enzyme was purified from the mitochondrial fraction of mouse liver to homogeneity as judged by polyacrylamide disc gel electrophoresis in the presence of dodecylsulphate. With histidine as amino donor, the enzyme was active with pyruvate, oxaloacetate and hydroxypyruvate as amino acceptors but not with 2-oxoglutarate. Effective amino donors were histidine, phenylalanine and tyrosine with pyruvate, and methionine, serine and glutamine with phenylpyruvate. The apparent Km for histidine was about 6.9 mM with pyruvate and that for pyruvate was 21 mM with histidine. The enzyme is probably composed of two identical subunits with a molecular weight of approximately 40000. The pH optimum was near 9.0. Isoelectric focusing of the purified enzyme resulted in the detection of four forms with pI 6.0, 6.2, 6.5 and 6.7, respectively, all of which were responsive to glucagon. These four forms were nearly identical with the purified enzyme before the focusing with respect to physical and enzymic properties. A possible mechanism of this multiplicity is discussed.


Catecholamines, adrenal hormones, and stress. From its beginnings about a century ago, research into the mechanisms underlying the stress response has led by many avenues to the adrenal medulla and its chromaffin cells. The latter possess cholinergic synapses that, when activated by a wide variety of stimuli, trigger discharge of catecholamines directly into the blood. Control of the response is mediated by a final common pathway arising in the hypothalamus and/or midbrain.


Atmospheric stability in cell culture vessels. We have investigated that atmospheric stability in polystyrene and glass cell culture vessels by measuring the dissolved O2 and CO2 in the media of both seeded and unseeded culture vessels incubated at 37 degrees C. There was no diffusion of either O2 or CO2 through glass vessels. At low partial pressures of oxygen (PO2), oxygen diffused into the polystyrene flasks at a rate of 1 to 2 mmHg per 24 hr, and at high PO2, oxygen diffused slowly out of polystyrene flasks. CO2 diffused out of polystyrene flasks with a half-time of 260 hr resulting in a considerable elevation in pH. In seeded polystyrene flasks with the PO2 less than or equal to room air, cellular oxygen consumption was masked by the inward diffusion of oxygen. In addition, the fall in pH due to metabolic CO2 and organic acid production during cell growth in polystyrene flasks was buffered by the diffusion of CO2 out of the vessels.


Culture of fetal mouse liver in plastic chambers: a new technique for organ culture. A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed in gas permeable roller tubes and rotated at 0.1 rpm in a CO2-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the cultivated liver. TAT activity was further induced by prednisolone. These results indicate that potential of this culture method for the study of physiological and pathological processes.


The antihypertensive effect of lexotan (bromazepam)- a new benzodiazepine derivative. Clinical observations led to the assumption that there is an antihypertensive effect of bromazepam in patients suffering from mild benign hypertension. Because of this observation we studied the antihypertensive effect of bromazepam by means of a standardized simple submaximal ergometric load performed weekly over a 4-week period of observation. A control group of 68 hypertensive patients without any drug therapy but under active physical training combined with physiotherapy and medicinal baths showed only a slight decrease in systolic and diastolic blood pressure at rest (about 4%). Furthermore, the pulse frequency did not change. In contrast, there was a distinct and significant decrease in blood pressure at rest and during exercise after a 3-week period of additional treatment with bromazepam, especially in hypertensive patients. One group of 68 hypertensive patients receiving 9 mg bromazepam daily showed a mean reduction in blood pressure by 14.4% systolic and 12.7% diastolic at rest, and during exercise by 7.5% systolic and 6.8% diastolic. The heart remained practically unchanged. A somewhat slighter decrease of the blood pressure values was seen in an additional group of 31 hypertensive patients receiving 6 mg bromazepam per day and in a group of 30 normotensive patients receiving 9 mg bromazepam daily. The calculated indices such as the product of heart rate and mean systolic pressure and the tension time index in the groups receiving bromazepam pointed to a better economic work performance under reduced myocardial pressure effort and reduced oxygen demand on the myocardium. The possible action of bromazepam in reducing blood pressure will be discussed.


Studies on groundnut proteins. VI. Isolation, characterisation and hydrogen ion titration of conarachin II. A method is described for isolating conarachin II in a homogeneous form by the techniques of DEAE-cellulose chromatography, polyacrylamide gel electrophoresis and sedimentation velocity. The protein contains 0.72% carbohydrate and no phosphate. Hydrogen ion titration curve indicated that the sidechain carboxyl, imidazol and epsilon-amino groups titrated with normal pK Int values and their number agreed with the analytical values obtained from amino acid analysis. However, tyrosine phenolic groups had abnormal pK Int of 10.5.


Reaction of bovine and equine growth hormones with tetranitromethane. Bovine and equine growth hormones were chemically modified with tetranitromethane, at pH 7.4 during 5 h and at pH 8.0 in the presence of 8 M urea during 1 h. a) Both hormones have very similar but not identical reactivities. b) The nitration of the reactive tyrosines and tryptophan residues at pH 7.4 produces no detectable changes in their immunological or somatotrophic activities. C) The nitration of all tyrosine residues in both hormones gives rise to a complete loss of somatographic activity with no alteration of the immunological activity.


The secondary structure of histone IV and its stability. The secondary structure within histone IV and its fragments obtained by cyanogen bromide (CNBr) and cleavage at Met 84 has been examined by circular dichroism and spectophotometric pH titration measurements. These studies have confirmed the existence of stable secondary structure within the C-terminal fragment of histone IV (C-peptide which can be perturbed only by 6M urea at pH greater than 8 or 8 M guanidine-HCL. In contrast, the N-terminal fragment (N-peptide) appears to lack significant secondary structure at low ionic strengths but acquires approximately 15% betasheet conformation and 5% alpha-helix upon aggregation at ionic strengths larger than or equal to 0.4. The rates of nitration of the N- and C-peptides by tetranitromethane (TNM) have also been measured as a function of ionic strengths. Under comparable conditions, the rate constant for nitration of the N-peptide was found to be about six times greater than that for the C-peptide, further evidence in support of the presence of stable secondary structure within the C-terminal region of histone IV. After binding these histone IV fragments to DNA, however, the nitration reaction rate constants for the N- and C-peptide in the bound form are found to be 2% and 27% of the corresponding free peptides. Reconstituted nucleohistone IV is about 10% as reactive to TNM as histone IV at comparable ionic strength.


Effect of clamping of the renal vein in dogs on certain biochemical and histopathological changes. Experimental investigations were carried out on dogs in which the renal vein was clamped for a period of 15 min, 30, and 60 min consecutively. It has been found that clamping of the renal vein for 15 min results in tissue anoxia and degeneration of the epithelium of the renal tubules. Clamping for 60 min causes cellular acidosis and necrosis of the tubules. It can therefore be said that clamping of the renal vein for even a short time is highly dangerous.


Gastric secretion.

Gastric secretion. This review has attempted to cover some of the findings that have been made in the mechanism of gastric secretion in recent years. It is hard to offer any firm conclusions, whether at the level of stimulus, metabolism, or the terminal process of secretion. However, some generalizations may be possible. At least amphibian gastric secretion is stimulated by cAMP as a second messenger, with histamine presumably acting as the primary messenger. The resultant metabolic change is due largely to a direct stimulation of catabolism, which in dog appears to be the metabolism of hexose, through the glycolytic process, the hexose monophosphate shunt, and the Krebs' cycle with cytoplasmic reduction and mitochondrial oxidation of pyridine nucleotides. No evidence could be obtained for changes in high energy phosphate or for lipolysis. One would expect gastric mucosal membranes during secretion to contain an anion-restricted electrogenic H+ pump, but they in fact contain an ATPase stimulated by monovalent cations and are insensitive to ouabain. In addition, hog or dog gastric membranes have the vectorial properties of H+ absorption, Rb+ extrusion, and ANS fluorescence enhancement with the addition of ATP, as well as protein phosphorylation by 32P dependent on a K+ gradient.


Cellular immune competence in patients with malignant tumors of the gastrointestinal tract. The local graft-vs-host reaction (GVHR) test was used to assess the cellular immune competence of lymphocytes obtained from 29 patients with malignant tumors of the gastrointestinal tract before and after removal of the tumor. Prior to removal most patients showed impairment of cellular immune competence; 10 to 14 days after surgery there was an improvement in a considerable number of patients. However, when tested four months later, the GVHR was again negative in some patients. The possible factors leading to deficiency in cellular immune competence and the value of the local GVHR test in the long-term follow-up of patients who have had malignant disease are discussed.


The impact of a physician assistant in diagnostic radiology [PA-DR] on the delivery of diagnostic radiologic clincial services. A program to train highly selected, experienced radiologic technologists to become Physician Assistants in Diagnostic Radiology [PA-DRs] was initiated at the University of Kentucky Medical Center in 1970. The initial postgraduate year of employment in several different radiologic environments for the first twelve trainees has been evaluated. Their work activities, including examinations that otherwise would have been performed by a radiologist and the number of radiographs screened for evidence of diseases, were analyzed. Studies during their training show they perform these activities accurately. Lastly, the economic impact of PA-DRs and their acceptability to patients and other professionals were evaluated. The PA-DRs spent a majority of their time in activities for which they were trained. Calculated yearly "earnings" of the PA-DRs averaged +31,164. Their salaries ranged +18-25,000. Each PA-DR averaged saving 34% of the employing radiologist's time. They were well accepted by professional colleagues and patients.


The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells. The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback. Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase was determined using H2O2 generated by the enzyme, D-alanine as a substrate, and cerous ions for the formation of an electron-dense precipitate. Both enzymes appeared to be located in microbodies. The combined presence of these enzymes characterizes the microbodies as peroxisomes. Biochemically and cytochemically, no urate oxidase or glycolate-oxidizing L-alpha-hydroxy acid oxidase could be demonstrated. Stereological analysis of the epithelia lining the renal tubules showed that the fractional volume of the microbodies is 5 to 10 times higher in the cells of the second proximal tubules than in the other nephronic segments or the ureter. The fractional volume of the microbodies was similar in kidneys of freshwater and seawater fishes.


Sampling and storage of blood for pH and blood gas analysis. Techniques used in sampling and storage of a blood sample for pH and gas measurements can have an important effect on the measured values. Observation of these techniques and principles will minimize in vitro alteration of the pH and blood gas values. To consider that a significant change has occurred in a pH or blood gas measurement from previous values, the change must exceed 0.015 for pH, 3 mm Hg for PCO2, 5 mm Hg for PO2, and 2 mEq/L for [HCO-3] or base excess/deficit. In vitro dilution of the blood sample with anticoagulant should be avoided because it will alter the measured PCO2 and base excess/deficit values. Arterial samples should be collected for meaningful pH and blood gas values. Central venous and free-flowing capillary blood can be used for screening procedures in normal patients but are subject to considerable error. A blood sample can be stored for up to 30 minutes at room temperature without significant change in acid-base values but only up to 12 minutes before significant changes occur in PO2. A blood sample can be stored for up to 3.5 hours in an ice-water bath without significant change in pH and for 6 hours without significant change in PCO2 or PO2. Variations of body temperatures from normal will cause a measurable change in pH and blood gas values when the blood is exposed to the normal water bath temperatures of the analyzer.


Chemical modification of coriolin B.

Chemical modification of coriolin B. Several derivatives of 5-ketocoriolin B (8, 11, 12) chemically modified at C-8 have been synthesized. These derivatives showed antitumor and antibacterial activity of the same degree as 5-ketocoriolin B (4) and diketocoriolin B (5) which were the most active members of the known coriolin group antibiotics. These derivatives were more stable than 4 and 5 in acidic or alkaline solution.


Causes of high blood O2 affinity of animals living at high altitude. We have measured the partial pressure of O2 at 50% saturation (P50) and the concentration of various phosphate compounds in the erythrocytes of the bar-headed goose and the guanaco to establish the cause of the high blood O2 affinity in animals who normally reside at high altitude. The same data were obtained in the blood of two goose species, that live at sea level, and in human blood. At standard conditions (pH 7.4, PCO2 40 Torr, 37 degrees C), P50 was 29.7 Torr in the blood of the bar-headed goose and was about 10 Torr higher in the goose species living at sea level. Since the concentration of organic phosphates was not markedly different in the erythrocytes of either goose species we conclude that the hemoglobin of the bar-headed goose reacts more weakly with organic phosphates, which can also be inferred from studies on purified hemoglobin solutions. Likewise, the low P50 of guanaco blood in comparison with human blood can be explained by a reduced interaction of 2,3-bisphosphoglycerate of guanaco hemoglobin compared to the human pigment.


Oxygen delivery and uptake in dogs during anemic and hypoxic hypoxia. Three groups of dogs were anesthetized, paralyzed, and ventilated at constant rates with the spleen clamped. Two groups were isovolemically hemodiluted with warm dextran and plasma to hematocrits just above and below that at which O2 uptake (VO2) could not be maintained at preanemic levels. One of these groups was given propranolol to reduce the cardiac output response to anemia. The third group was ventilated on a low O2 gas mixture to decrease oxygen uptake. VO2 was thus limited at a high-delivery O2 pressure (PO2) in anemia and a low-delivery PO2 in hypoxic hypoxia. VO2 was reduced at a mixed venous PO2 of 45 Torr in anemia and at 17 Torr in hypoxic hypoxia. VO2, mixed venous PO2, and O2 delivery decreased precipitously at hematocrits below 10%. Once VO2 was limited by O2 availability, a single linear relationship (r = 0.91) was found for percent VO2 as a function of total O2 delivery (cardiac output X arterial O2 content) during both anemic and hypoxic hypoxia. The critical value for O2 delivery was 9.8 ml/kg-min. When O2 supply became limiting, VO2 apparently was not diffusion limited because it was more dependent on volume delivery rates than on delivery PO2.


Can alveolar pCO2 exceed pulmonary end-capillary CO2? Yes. In 1891 Christian Bohr observed that alveolar PCO2 could exceed arterial PCO2 when high levels of CO2 were present in the inspired air. He proposed that CO2 could be secreted by the lung. His hypothesis was dismissed as being due to experimental error; however, recently the same phenomenon was rediscovered using modern techniques. A possible explanation for the phenomenon may involve coupling between diffusion and chemical reaction of H+, HCO3-, and CO2 in the vicinity of a negatively charged capillary wall, causing the PCO2 to be higher near the wall that in the bulk phase of capillary blood.  Alveolar PCO2 is related to the PCO2 near the wall rather than the bulk phase PCO2. The Charged Membrane Hypotheses can explain the phenomenon of CO2 secretion not by a cellular mechanism, but a physical chemical disequilibrium within the capillary which is maintained by blood flow through the capillary.


Can alveolar pCO2 exceed pulmonary end-capillary CO2? No. The exchanges and chemical reactions of CO2 within the blood are not complete during its transit time through the alveolar capillaries, so that theoretically alveolar PCO2 cannot exactly equal end-capillary blood PCO2. However, as a practical matter, the technical errors in determining alveolar and peripheral arterial PCO2 are so large in comparison with the difference between pulmonary arterial PCO2 and pulmonary venous PCO2, owing to the large effective solubility of CO2 in blood, that for this reason alone any difference between alveolar and end-capillary PCO2 can be neglected. The hypothetical mechanism that was originally proposed by Gurtner, Song, and Farhi for the production of an alveolar PCO2 greater than end-capillary PCO2 during rebreathing and depends on the existence of a radial gradient of H+ X HCO3- in the capillary blood near the wall appears unlikely because a) it would require a significant separation of charged ions over a large proportion of the capillary length and for a large proportion of the blood transit time and b) it would require a large radial gradient of PCO2 within the blood which on thermodynamic grounds would in turn require an unreasonable amount of energy to maintain.


Pulmonary vascular response to increase in intracranial pressure: role of sympathetic mechanisms. The pulmonary vascular response to intracranial hypertension was studied in anesthetized controlled ventilated dogs in which intracranial pressure (ICP) was elevated to 20 Torr below the mean arterial pressure for a 20-min period, and regulated at this level. Pulmonary vascular resistance (PVR) increased from control value of 2.7 +/- 0.30 to 8.3 +/- 0.51 Torr-l-1-min at the end of 20-min increase in ICP. The increase in PVR was associated with marked increase (P less than 0.001) in pulmonary arterial pressure from 14.4 +/- 1.3 to 35.4 +/- 4.0 Torr, small increase in left atrial pressure from 5.4 +/- 1.2 to 7.9 +/- 1.9 Torr, and no significant change in pulmonary blood flow. The increase in PVR occurred independently of changes in the arterial pressure. The increase in PVR induced by elevated ICP was correlated with increases in lung water, physiological shunt (Qs/Qt), alveolar dead space (VD), and with hypoxemia. Pretreatment with propranolol (1.5 mg-kg-1) attenuated the increase in PVR during elevation in ICP; the smaller increase in PVR was associated with a marked increase in left atrial pressure and a smaller increase in pulmonary perfusion pressure than in the control group. The propranolol-treated dogs also developed increases in lung water, Qs/Qt, VD, and hypoxemia. In contrast, pretreatment with phenoxybenzamine (1.5 mg-kg-1) inhibited the increases in pulmonary perfusion pressure and PVR induced by ICP elevation as well as the associated increases in lung water, Qs/Qt, VD, and hypoxemia. Therefore, a sustained elevation in ICP at a level below the mean arterial pressure in the intact dog evokes pulmonary vasoconstriction which is mediated by alpha-adrenergic mechanisms. The neurogenic pulmonary vasoconstriction results in the increases in lung water, Qs/Qt, VD, and in the hypoxemia.


Cardiovascular, respiratory, and metabolic adjustments to exercise in dogs. Cardiovascular and metabolic parameters were studied in dogs anesthetized with pentobarbital sodium, and while awake resting or exercising for 30 min at either 6.4 km/h, 10% grade (32% VO2 max) or 8.0 km/h, 16% grade (50% VO2 max). The anesthetized dogs had lower cardiac outputs, stroke volumes, arterial-mixed venous oxygen differences, oxygen uptakes, rectal temperatures, and higher diastolic and mean arterial pressures than awake resting dogs. Heart rates and arterial systolic pressures were similar in the two conditions. The increased oxygen uptakes during exercise were associated with approximately equal percentage increments in cardiac outputs and oxygen extractions. Cardiac output increases during exercise were largely due to increases in heart rates. Arterial CO2 tension and CO2 contents as well as venous O2 and CO2 gas tensions and contents declined, and pH and rectal temperatures increased during exercise. The dogs became alkalotic during exercise. Elevated central body temperatures appeared to be the major factor controlling respiration.


Effect of respiratory acidosis and activity on airway smooth muscle intracellular pH. Previous work in our laboratory has shown that respiratory acidosis (RA) impaired mechanical function in canine tracheal smooth muscle (TSM). Since an intracellular acidosis could be brought on by the increased CO2 content of the bathing medium and alter the Km's of rate-limiting glycolytic enzymes in the pathway of energy production for contractile function, we have investigated the effects of RA on the intracellular pH (pHi) of TSM. Using the DMO method, paired unstimulated or resting TSM strips were incubated under normocapnic conditions (PO2 600 Torr, PCO2 40 Torr, pH 7.40) and RA (PO2 550 Torr, PCO2 110 Torr, pH 6.95) with 14C-labeled DMO and 3H-labeled inulin or PEG-4000. In another set of paired experiments, TSM strips were tetanized electrically every 5 min or pharmacologically throughout the incubation period ("active" muscle strips). The tissue and an aliquot of bathing medium were counted for 3H and 14C content and the values entered into the Wadell and Butler equation. The pHi's of "resting" normocapnic and acidotic strips were 7.041 +/- 0.017 (SE) and 6.752 +/- 0.012, respectively. However, the pHi's of "active" normocapnic and acidotic strips were 7.275 +/- 0.017 and 7.017 +/- 0.015, respectively. We conclude that respiratory acidosis lowers intracellular pH in both resting and mechanically active TSM's; however, "active" preparations whether exposed to normocapnia or acidosis were unexpectedly more alkaline than their "resting" counterparts.


Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.


Isolation and properties of a thermostable restriction endonuclease (ENDO R-Bst1503). A restriction endonuclease was isolated from Bacillus stearothermophilus1503-4R (Bst1503) and purified to homogeneity. The enzyme required Mg2+ ion as a cofactor. Bst1503 exhibited maximal activity between pH 7.5 and 8.0, between 60 and 65 degrees C, and with about 0.2 mM Mg2+. Bst1503 was not inactivated after exposure at 55 or 65 degrees C for up to 10 h. After 2 h of incubation at 70 degrees C, Bst1503 was inactivated by 65%. Bst1503 was rapidly inactivated at 75 degrees C. A single protein-staining band having a molecular weight of 46,000 was observed when Bst1503 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to exist in two active forms, the predominating form with an S value of 8.3 (180,000) and the second form with an S value of 5.4 (96,000). No conversion between the 8.3S and 5.4S forms was observed after storage. Bst1503 recognized six sites in TP-1C deoxyribonucleic acid (DNA), one site in pSC101 and simian virus 40 DNAs, and three sites in lambdavir DNA. Bst1503 and BamHI were determined to be isoschizomers. The effect of temperatures on the activity and stability of BamHI was determined.


Repression of the tyrosine, lysine, and methionine biosynthetic pathways in a hisT mutant of Salmonella typhimurium. A comparison was made of the repressibility of certain enzymes in the tyrosine, methionine, and lysine biosynthetic pathways in wild-type Salmonella typhimurium and a hisT mutant. The results show that (i) tyrosine represses the synthesis of the tyrosine-sensitive 3-deoxy-D-arabino-heptulsonic acid 7-phosphate synthetase and the tyrosine aminotransferase to the same extent in a hisT mutant as in wild type and (ii) there is no detectable alteration in the extent to which methionine represses O-succinylhomoserine synthetase or in the extent to which lysine represses the lysine-sensitive beta-aspartokinase as a result of the hisT mutation.


Killer toxin for sake yeast: properties and effects of adenosine 5'-diphosphate and calcium ion on killing action. The killer character of strain isolated from the main mash of sake brewing which produces a killer substance for sake yeast was transmitted to hybrids of the strain and a standard strain of Saccharomyces cerevisiae through a cytoplasmic determinant. The character was eliminated at 41 degrees C by incubation followed by growth at 30 degrees C. The killer strain produced the killer toxin in a growth-associated manner. A preparation of crude killer toxin extract showed first-order inactivation and a linear Arrhenius plot between 25 and 40 degrees C, with an activation of energy of 55.0 kcal/mol. Addition of 1% of synthetic polymer protected the toxin from inactivation by agitation but not by heat. Enhancement of the killer action toward sensitive yeast cells by only the nucleotide adenosine 5'-diphosphate (ADP) was observed after plating on agar medium as well as after incubation in liquid medium. The addition of CaCl2 reversed the enhancing effect of ADP on killing activity. This action of CaCl2 was inhibited by cycloheximide, suggesting that protein synthesis is required for recovery of toxin-induced cells in the presence of CaCl2. Further, CaCl2 overcame the decrease in the intracellular level of adenosine 5'-triphosphate (ATP) enhanced by ADP in killer-treated cells and also inhibited leakage of ATP from the cells with immediate response. The mode of killing action is discussed in terms of a transient state of the cells and the action of ADP and CaCl2.


Membrane-bound thioesterase activity in mycoplasmas. Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity of Acholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 18 degrees C, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibllity that the activity of the enzyme is controlled by the physical state of membrane lipids are discussed.


Characterization of autolysins from Mycobacterium smegmatis. This study demonstrates, for the first time, the autolytic enzymes associated with mycobacterial cell walls. Based on the release of radioactivity and ninhydrin-reactive material from isolated cell walls, it was shown that maximum activity occurs during the late log phase of growth and at a buffer pH of about 8.0. Chemical analyses of autolytic digests of isolated cell walls indicated that at least three autolysins are active under the conditions used. These are N-glycolylmuramic acid-L-alanine amidase, an aminopeptidase that releases L-alanine, and an endopeptidase that solubilizes and L-alanyl-D-glutamic acid dippetide. No other endopeptidase, carboxypeptidase, or glycosidase activity was detected.


Adenosine 5'-triphosphate synthesis driven by a protonmotive force in membrane vesicles of Escherichia coli. Adenosine 5'-triphosphate (ATP) synthesis energized by an artificially imposed protonmotive force (delta p) in adenosine 5'-diphosphate-loaded membrane vesicles of Escherichia coli was investigated. The protonmotive force is composed of an artificially imposed pH gradient (delta pH) or membrane potential (deltapsi), or both. A delta pH was established by a rapid alteration of the pH of the assay medium. A delta psi was created by the establishment of diffusion potential of K+ in the presence of valinomycin. The maximal amount of ATP synthesized was 0.4 to 0.5 nmol/mg of membrane protein when energized by a delta pH and 0.2 to 0.3 nmol/mg of membrane protein when a delta psi was imposed. Simultaneous imposition of both a delta pH and delta psi resulted in the formation of greater amounts of ATP (0.8 nmol/mg of membrane protein) than with either alone. The amount of ATP synthesized was roughly proportional to the magnitude of the artificially imposed delta p. Although p-chloromercuribenzoate, 2-heptyl-4-hydroxyquinoline-N-oxide, or NaCN each inhibits oxidation of D-lactate, and thus oxidative phosphorylation, none inhibited ATP synthesis driven by an artificially imposed delta p. Membrane vesicles prepared from uncA or uncB strains, which are defective in oxidative phosphorylation, likewise were unable to catalyze ATP synthesis when energy was supplied by an artificially imposed delta p.


Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.


Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase. An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.


Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium. Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.


Regulation of glnA messinger ribonucleic acid synthesis in Klebsiella aerogenes. We examined wild-type and mutant strains of Klebsiella aerogenes for the relative amounts of ribonucleic acid (RNA) hybridizing specifically to deoxyribonucleic acid from a transducing phage carrying glnAK, the structural gene for glutamine synthetase. Our data showed a positive correlation between the intracellular level of glutamine synthetase and the level of glnA messenger RNA; we were unable to detect glnA messinger RNA in strains devoid of glutamine synthetase protein. Therefore, it is possible that transcription of glnA is not regulated simply by repression mediated through the glutamine synthetase protein; rather, autogenous control in this system may involve activation of transcription. Our experiments also suggest that the promotor of the glnA gene is located at the rha proximal end of the gene.


Isolation of an Escherichia coli mutant deficient in thioredoxin reductase. A mutant of Escherichia coli defective in thioredoxin reductase has been isolated and partially characterized. This mutant has no detectable thioredoxin reductase activity in vitro and yet it exhibits no in vivo defect in reduction of ribonucleotides. Evidence is presented that indicates that, in cells permeabilized via ether treatment, ribonucleoside diphosphate reduction can utilize glutathione as an alternate reducing system.


Factors affecting genetic transformation of Neisseria gonorrhoeae. Piliated gonococci were competent in genetic transformation in all stages of growth in minimal and enriched media, but nonpiliated cells were almost totally incompetent. Uptake of deoxyribonucleic acid into a deoxyribonuclease-insensitive state was observed only in competent piliated cells. Competence was not affected by washing of competent cells or treatment of competent cells with proteolytic enzymes. Expression of competence required presence of any of several different monovalent or divalent cations, as well as a utilizable source of energy. Efforts to produce genotypically or phenotypically competent derivatives of nonpiliated cells were unsuccessful. These experiments are consistent with the idea that pili may play a role in the irreversible uptake of transforming deoxyribonucleic acid by the gonococcus, but fail to provide evidence for other types of competence factors.


Glutamine synthetase of Klebsiella aerogenes: genetic and physiological properties of mutants in the adenylylation system. Mutations resulting in defects in the adenylylation system of glutamine synthetase (GS) affect the expression of glnA, the structural gene for GS. Mutants with lesions in glnB are glutamine auxotrophs and contain repressed levels of highly adenylylated GS. Glutamine-independent revertants of the glnB3 mutant have acquired an additional mutation at the glnE site. The glnE54 mutant is incapable of adenylylating GS and produces high levels of enzyme, even when ammonia is present in the growth medium. The fact that mutations in glnB and glnE simultaneously disturb both the normal adenylylation and repression patterns of GS in Klebsiella aerogenes indicates that the adenylylation system, or adenylylation state, of GS is critical for the regulation of synthesis of GS.


Characterization of beta-galactosidase from a special strain of Aspergillus oryzae. beta-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from alpha-galactosidase, alpha- and beta-mannosidase, alpha- and beta-N-acetylhexosaminidase, and protease activities. The beta-galactosidase was capable of acting on aryl beta-galactosides, lactose, and lactosides. It also hydrolyzed beta-galactosyl linkages in urinary glycoasparagines and asialo alpha1-acid glycoprotein. The enzyme was rather stable in aqueous solution, retaining full activity at 4 degrees for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37 degrees, full activity was maintained for several days.


The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate. 1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxymethyl rho-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the gamma-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent. 2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly histidine residue(s)). 3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts. 4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.


The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide. 1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.


Dog renal kallikrein: purification and some properties. The contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2,000-fold with an overall yield of 18% by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50. The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per A280, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8 X 10(4) from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3. The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein.


The reactive site of eggplant trypsin inhibitor. The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.


A study of the native-denatured (N in equilibrium with D) transition in lysozyme. III. Effect of alteration of net charge by acetylation. Measurement of the enzymic activity and fluorescence properties showed that the gross conformation of acetylated lysozyme [EC 3.2.1.17] is very similar to that of the native enzyme. On the other hand, protease digestion, t-butyl hypochloride modification and thermal denaturation experiments performed on native, acetylated, and guanidinated lysozymes showed that acetylation caused a small but significant shift of the N in equilibrium with D transition to the right. Thus it can be concluded that charge balance in a protein plays an important role in maintaining its conformation. The difference between equilibrium and kinetic methods of monitoring protein denaturation was also clarified.


Exchange reactions catalyzed by methioninase from Pseudomonas putida. Isolation and characterization of the exchange products. Gel-electrophoretically homogeneous methioninase [L-methionine methanethiol-lyase (deaminating), EC 4.4.1.11] of Pseudomonas putida, which catalyzes alpha, beta- and alpha, gamma-eliminations from S-substituted amino acids, could also catalyze a variety of beta- and gamma-exchange reactions, according to the following equations: RSCH2CH(NH2)COOH+R'SH in equilibrium R'SCH2CH(NH2)COOH+RSH (beta-exchange) and RSCH2CH2CH(NH2)COOH+R'SH in equilibrium R'SCH2CH2CH(NH2)COOH+RSH (gamma-exchange), where R'SH represents an exogeneously added alkanethiol or a substituted thiol. Related amino acids not available for elimination reactions appeared to be inert as substrates for exchange. The maximum activity for the exchange reactions was observed at pH 8.5 in potassium pyrophosphate buffer. The activity increased linearly with the increase in protein concentration from zero to 3.0 mug per ml, and with incubation time up to at least 15 min at 30 degrees. Some of the exchange reaction products were purified by a combination of paper and ion exchange chromatographies, and charcoal treatment: their structures were confirmed by physicochemical methods including elemental analysis and proton magnetic resonance, infrared, and mass spectrometries.


Determination of the intravesicular pH of fragmented sarcoplasmic reticulum with 5,5-dimethyl-2,4-oxazolidinedione. The intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56. It was also demonstrated by direct measurements of pHe with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions. From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively.


Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12. The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 mM Mg2+. A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60 degrees. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8-O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.


A novel neutral protease(s) from monkey liver. A novel neutral protease(s), which is presumably membrane-bound, was found in monkey liver using heat-denatured casein as a substrate and was separated from other major catheptic proteases by successive procedures of gel filtration on Ultrogel AcA 22, solubilization by deoxycholate and gel filtration on Sepharose 6B. The enzyme(s) showed maximal activity at pH 8.0, and was strongly inhibited by DFP and PMSF. Many other reagents tested, including TPCK, TLCK, pCMB, iodoacetic acid, and EDTA, were without marked effect on the activity. Activation of the enzyme(s) by NaCl was not observed.


Control of 3-hydroxy-3-methylglutaryl coenzyme A reductase by endogenously synthesized sterols in vitro and in vivo. Isolated rat hepatocytes converted mevalonolactone into sterol intermediates and fatty acids 6- to 8-fold faster than mevalonate salt at concentrations less than 6 X 10(-4) M. Incubation of hepatocytes for 3 h normally results in induction of 3-hydroxy-3-methylglutaryl-CoA reductase. This increase in enzyme activity was inhibited by mevalonolactone and by mevalonate salt; at each concentration between 6 X 10(-4) M and 6 X 10(-8) M the lactone was a more effective inhibitor than the salt. The increase in enzyme activity was completely prevented by 6 X 10(-4) M lactone, and at this concentration the cells synthesized from the lactone an amount of sterol per hour which approximated that leavingthe cells in the same period. Administration of mevalonolactone to intact rats resulted in a dose-dependent inhibition of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. At the highest dose (400 mg of (RS)-mevalonolactone/200 g of rat) enzyme activities declined 85% within 45 min and were still suppressed below normals after 28 h. Mevalonolactone treatment resulted in increases in liver cholesterol content and in the cholesterol ester concentration of liver microsomes. The results demonstrate that the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase can be controlled by the rate of endogenous sterol synthesis both in vitro and in vivo.


Binding of tritiated bovine parathyroid hormone to plasma membranes from bovine kidney cortex. A membrane fraction enriched in parathyroid hormone (PTH)-sensitive adenylate cyclase and sodium and potassium ion-activated (Na+, K+)-ATPase was prepared from bovine kidney. Tritiated PTH binding to this membrane fraction was dependent on both hormone and membrane protein concentration. Both total and specific binding of the hormone decreased significantly after 5 to 10 min of incubation at 22 degrees. PTH binding was highly specific, being sensitive to inhibition only with active forms of unlabeled hormone (native and 1-34 PTH). Specific binding showed a pH optimum of 7.3 to 7.5. Inhibition of binding of tritiated hormone by unlabeled PTH was also highly effective at pH 6.0, but this apparently specific binding was also inhibited by adrenocorticotropic hormone, insulin, glucagon, and vasopressin. Dissociation of bound hormone was demonstrated, and an apparent dissociation constant of 4.6 X 10(-2) min-1 was obtained. Specific binding was eliminated by pretreatment of the membranes with trypsin. The concentration dependence for inhibition of binding with unlabeled PTH was identical to that for activation of adenylate cyclase in this membrane preparation, and binding was also inhibited by concentrations of calcium in the 0.5 to 2 mM range.


Studies of binding of parathyroid hormone to a detergent-dispersed preparation from bovine kidney cortex plasma membranes. Bovine kidney plasma membranes containing parathyroid hormone-sensitive adenylate cyclase activity were dispersed with 1% Triton X-100 and centrifuged at 150,000 X g for 2 h. Approximately 40% of the total membrane protein was extracted by this procedure. The extraction greatly reduces the fluoride-stimulated and the parathyroid hormone-sensitive adenylate cyclase activity of the membranes and yields a supernatnat which binds biologically active, tritiated parathyroid hormone. Hormone binding is stable for up to 15 h and has a linear dependence on protein concentration in the extract. Binding of the labeled hormone at concentrations of 5 to 10 nM is inhibited by preincubation with unlabeled min, and displays a dependence on temperature, time, and pH. Binding specificity is maximal at physiological pH, being inhibited by only the native hormone or its synthetic 1-34 NH2-terminal, biologically active fragment. Binding increases dramatically at pH 6.0, but is nonspecific in character. Half-maximal inhibition of the binding was achieved at 3.2 X 10(-7) M concentrations of the native hormone and 5.0 X 10(-7) M concentrations of the synthetic 1-34 NH2-terminal fragment. Calcium does not inhibit either total or specific binding. Inhibition, kinetic, and pH dependence data suggest that the extracted component(s) represent the parathyroid hormone binding protein(s) formerly identified in particulate membrane preparations.


Binding of nucleotides to purified coupling factor-latent ATPase from Mycobacterium phlei. Binding studies of various nucleotides to the purified coupling factor-latent ATPase from Mycobacterium phlei have been carried out using gel filtration, equilibrium dialysis, and ultrafiltration methods. The purified latent ATPase binds 3 mol of ADP per mol of the enzyme with an apparent dissociation constant of 68 muM. Binding of nucleotides occurred in the decreasing order: ADP, epsilon-ATP, epsilon-ADP, UDP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), IDP, and adenosine 5'-(alpha,beta-methylene)diphosphate (AdoP(CH2)P). AMP-P(NH)P inhibits both soluble (Ki = 77 muM) and membrane-bound latent ATPase activity. However, AMP-P(NH)P does not affect oxidative phosphorylation in membrane vesicles of M. phlei. AMP-P(NH)P exhibits one binding site per molecule of the enzyme with a dissociation constant of 71 muM. After trypsin treatment of the enzyme, the binding of ADP decreases 35%, while AMP-P(NH)P binding remains unchanged. Moreover, AMP-P(NH)P binding was not displaced by ADP. Studies with sulfhydryl agents showed that, in contrast to AMP-P(NH)P, binding of at least 1 mol of ADP requires the participation of sulfhydryl groups. The results indicate that AMP-P(NH)P and ADP do not share a common binding site and that the latent ATPase enzyme has separate sites for ATP hydrolysis and ATP synthesis.


Protein kinase activity at the inner membrane of mammalian mitochondria. This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane.


Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base. Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.


beta-Glucoside hydrolase activity of normal and glucosylceramidotic cultured human skin fibroblasts. Cultured human skin fibroblasts from normal and glucosylceramidotic subjects are found to contain one beta-glucoside hydrolase as compared with multiple enzymes in other tissues. The fibroblast enzyme has an approximate molecular weight of 150,000 under isotonic conditions, as determined by gel filtration. It occurs as a large aggregate at low ionic strength. Ceramide, 4-methylumbelliferyl, and p-nitrophenyl beta-glucosides are active as substrates. The enzyme in whole cell homogenates is membrane-bound and is solubilized by a combination of Triton X-100 and sodium taurocholate. It has a pH optimum at 4.2 and no demonstrable divalent cation requirement. The cultured fibroblast beta-glucosidase displays close similarity to one of the forms of beta-glucosidase in human spleen, specifically that form which is affected in Gaucher's disease. 4-Methylumbelliferyl beta-glucosidase activity in homozygous fibroblasts from infantile and adult forms of Gaucher's disease are reduced to 9 and 14%, respectively, of normal fibroblast activity. The residual activity in the lipidotic cells shows increased heat lability, but cannot be distinguished from that in normal cells with respect to gel exclusion properties, Michaelis constant, and pH dependence.


Transport and metabolism of vitamin B6 in lactic acid bacteria. Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.


Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase. The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.


Solubility and diffusion coefficient of adenosine 3':5'-monophosphate. Several previously unavailable parameters of adenosine 3':5'-monophosphate have been determined. The molar extinction coefficient at pH 7.0 is 1.38 X 10(-4), the aqueous solubility at pH 7.0 is 0.0236 M and the diffusion coefficient is 4.44 X 10(-6) cm2/s.


Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase. 1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.


Properties of passive binding of calcium to endoplasmic reticulum from adipocytes. Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. Binding was concentration-dependent, saturable, and totally dissociable. Steady state was reached within 20 min at all calcium concentrations tested. Three apparent classes of binding sites were identified in kinetic and steady state studies using calcium concentrations from 1 muM to 10 mM. The affinity constants (and maximum binding capacities) as determined by computer analysis for the three classes were 2.1 X 10(5) M-1 (0.28 nmol of calcium/mg of protein), 1.3 X 10(4) M-1 (1.1 nmol/mg), and 1.3 X 10(2) M-1 (35 nmol/mg). The dissociation rate constants for the high and intermediate affinity classes of sites were 1.6 X 10(-3) S-1, respectively, and the association rate constant for the high affinity sites was 8 X 10(2) M-1 S-1. The affinity constant calculated from the rate constants was 5.0 X 10(5) M-1 for the high affinity sites in agreement with the value obtained in studies at steady state. The three classes of binding sites were specific for calcium. Magnesium was a noncompetitive inhibitor of calcium binding to all three classes of sites with a Ki of 9 to 12 mM. Calcium binding at 1 muM calcium was 50% inhibited by 18 muM La3+, 600 muM Sr2+, or 2.7 mM Ba2+. These data represent the first analysis of passive calcium binding to endoplasmic reticulum from nonmuscular cells and the first report of corresponding rate constants for either endoplasmic or sarcoplasmic reticulum. The characteristics of the binding are consistent with the properties of calcium transport by endoplasmic reticulum of adipocytes. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis.


Decreased lysyl oxidase activity in the aneurysm-prone, mottled mouse. Inbred mice bearing certain alleles at the Mottled locus have defects in connective tissue which result in weakness of skin and of blood vessels. Previous studies have established that cross-links in collagen and elastin are decreased in these animals due to impaired formation of lysine-derived aldehydes. Lysyl oxidase activity in extracts of skin is markedly lower in those prepared from affected animals than control mice. An inhibitor of lysyl oxidase is present in equal amounts in affected and control skins and does not account for diminished activity found in affected animals.


Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides. Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.


Proton inactivation of Ca2+ transport by sarcoplasmic reticulum. The effects of acid on fragmented sarcoplasmic reticulum from rabbit white skeletal muscle have been studied. Brief exposure of sarcoplasmic reticulum membranes to pH values in the range 5.5 to 6.0 at 37 degrees caused rapid inactivation of calcium accumulation measured at 25 degrees in the presence of oxalate (calcium uptake) while (Ca2+, Mg2+)-ATPase (EC 3.6.1.3) activity was enhanced by 75%. ATPase activity, measured at 37 degrees in the absence of oxalate and in the calcium steady state, was unaltered when calcium uptake was inactivated. Calcium efflux from sarcoplasmic reticulum vesicles, previously loaded passibely with 45CaCl2, was only slightly increased when calcium uptake was abolished. At still lower pH values, 5.0 to 5.5, (Ca2+, Mg2+)-ATPase was inactivated while Mg2+ ATPase was more acid-resistant. Acid inactivation of calcium uptake followed simple first order kinetics for at least 80% of the time course. The rate constant, k, increased from 0.043 min-1 to 1.63 min-1 between pH 6.50 and pH 5.35. At pH 4.65, Ea, the energy of activation, was 31 kcal mol-1 between 24 degrees and 43 degrees. Inactivation, once initiated, was irreversible. Aged suspensions of sarcoplasmic reticulum were more sensitive to acid inactivation. Ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid enhanced inactivation, and calcium specifically protected against inactivation with half-maximal effect at 1 to 2 mM. The sulfhydryl reagent, dithiothreitol (1 mM), caused significantly increased rates of inactivation. Calcium binding was studied by dual wavelength spectrophotometry and stopped flow analysis. Acid inactivation distinguished two ATP-induced binding sites, previously described (Entman, M. L., Snow, T. R., Freed, D., and Schwartz, A. (1973) J. Biol. Chem. 248, 7762-7772) as a superficial Mg2+-independent Site A which binds and releases calcium rapidly and a deeper Mg2+-dependent Site B which binds and releases calcium more slowly. Rates of binding to both sites were decreased by acid inactivation. Binding of calcium to Site A increased, however, from 4.6 to 6.4 nmol mg of protein-1 whereas that to Site B decreased from 17.0 to 6.9 nmol mg of protein-1. Passive binding of calcium to sites of medium affinity (K = 7 X 10(4) M-1) was unaffected by acid inactivation of calcium uptake. Temperature dependence of (Ca2+, Mg2+)-ATPase was unchanged in the range 9-34 degrees. Above 34 degrees, the higher activation energy process (Ealpha = 33.7 kcal mol-1) observed in control sarcoplasmic reticulum and thought to arise from a conformational change in the ATPase (Inesi, G., Millman, M., and Eletr, S. (1973) J. Mol. Biol. 81, 483-504) was diminished by acid inactivation (Ealpha = 8.2 kcal mol-1) in a manner suggesting that it is related to active calcium transport. The ATP in equilibrium 32Pi exchange reaction was diminished by acid, but 25% of the activity remained when calcium uptake was completely abolished...


Podophyllotoxin as a probe for the colchicine binding site of tubulin. The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.


Purification and properties of galactokinase from Saccharomyces cerevisiae. Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully induced for the production of the galactose metabolizing enzymes. The purification was accomplished by:(a) ammonium sulfate fractionation, (b) streptomycin sulfate precipitation. (c) DEAE-cellulose chromatography, (d) hydroxylapatite chromatography, and finally (e) Bio-Gel A-0.5 m gel filtration. The resulting preparation of galactokinase was judged to be at least 95% pure by the following criteria: (a) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) ultracentrifuge analysis, (c) nondissociating polyacrylamide gel electrophoresis, and (d) Bio-Gel A-0.5 m gel filtration. The purified enzyme preparation was used to determine the Km values for the two substrates, galactose and ATP, which were found to be 0.60 and 0.15 mM, respectively. Vmax was also determined and found to be 3.35 mmol/h/mg. This corresponds to a turnover rate of 3350 molecules of galactose phosphorylated/min/enzyme molecule. The effect of pH on the galactokinase-catalyzed phosphorylation of galactose was determined; the results showed the pH optimum of the reaction to be in the range of pH 8.0 to 9.0. The enzyme is highly specific for galactose since galactokinase did not appear to phosphorylate any of the other sugars tested at a rate greater than 0.5% of the rate of galactose phosphorylation. Amino acid analysis was performed on the enzyme preparation and the results were used to calculate the partial specific volume (v) of 0.736. The NH2-terminal sequence was determined for the first 3 residues. The molecular weight and subunit composition were determined by ultracentrifugation and polyacrylamide gel electrophoresis under dissociating and nondissociating conditions. The data obtained indicated that galactokinase is a monomeric protein of molecular weight 58,000.


Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation. Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.


Human endonuclease specific for apurinic/apyrimidinic sites in DNA. Partial purification and characterization of multiple forms from placenta. Six chromatographically distinct forms of endonuclease active on apurinic and apyrimidinic sites in DNA have been purified away from DNA phosphatases, DNA N-glycosidases, and other DNases of human placenta. The forms seem to be monomeric proteins of 27,000 to 31,000 daltons, and although catalytically similar, they can be distinguished from one another on the basis of substrate Km and the effects of small molecules such as ATP. Analysis of enzymatic activity on a spectrum of damaged DNA substrates indicates that the enzyme forms probably act at an appreciable rate only adjacent to the phosphodiester bond of a deoxyribose lacking a base (purine or pyrimidine) in duplex DNA; such sites can be formed by treating the DNA with acid, alkylating agents, DNA N-glycosidases, and, probably, x-rays and OsO4. The incision is made so as to form a deoxyribose 5'-phosphate and a 3'-hydroxynucleotide.


Effect of aspartate on complexes between glutamate dehydrogenase and various aminotransferases. In previous studies it was found that: (a) aspartate aminotransferase increases the aspartate dehydrogenase activity of glutamate dehydrogenase; (b) the pyridoxamine-P form of this aminotransferase can form an enzyme-enzyme complex with glutamate dehydrogenase; and (c) the pyridoxamine-P form can be dehydrogenated to the pyridoxal-P form by glutamate dehydrogenase. It was therefore concluded (Fahien, L.A., and Smith, S.E. (1974) J. Biol. Chem 249, 2696-2703) that in the aspartate dehydrogenase reaction, aspartate converts the aminotransferase into the pyridoxamine-P form which is then dehydrogenated by glutamate dehydrogenase. The present results support this mechanism and essentially exclude the possibility that aspartate actually reacts with glutamate dehydrogenase and the aminotransferase is an allosteric activator. Indeed, it was found that aspartate is actually an activator of the reaction between glutamate dehydrogenase and the pyridoxamine-P form of the aminotransferase. Aspartate also markedly activated the alanine dehydrogenase reaction catalyzed by glutamate dehydrogenase plus alanine aminotransferase and the ornithine dehydrogenase reaction catalyzed by ornithine aminotransferase plus glutamate dehydrogenase. In these latter two reactions, there is no significant conversion of aspartate to oxalecetate and other compounds tested (including oxalacetate) would not substitute for aspartate. Thus aspartate is apparently bound to glutamate dehydrogenase and this increases the reactivity of this enzyme with the pyridoxamine-P form of aminotransferases. This could be of physiological importance because aspartate enables the aspartate and ornithine dehydrogenase reactions to be catalyzed almost as rapidly by complexes between glutamate dehydrogenase and the appropriate mitochondrial aminotransferase in the absence of alpha-ketoglutarate as they are in the presence of this substrate. Furthermore, in the presence of aspartate, alpha-ketoglutarate can have little or no affect on these reactions. Consequently, in the mitochondria of some organs these reactions could be catalyzed exclusively by enzyme-enzyme complexes even in the presence of alpha-ketoglutarate. Rat liver glutamate dehydrogenase is essentially as active as thebovine liver enzyme with aminotransferases. Since the rat liver enzyme does not polymerize, this unambiguously demonstrates that monomeric forms of glutamate dehydrogenase can react with aminotransferases.


Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose. We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.


Translation of albumin messenger RNA in a cell-free protein-synthesizing system derived from wheat germ. Purified rat liver albumin mRNA directed the synthesis of albumin in a mRNA-dependent cell-free protein-synthesizing system derived from wheat germ extracts. The [3H]leucine-labeled in vitro translation product reacted with antibodies specific for albumin and co-migrated with authentic 14C-labeled serum albumin during gel electrophoresis in the presence or absence of sodium dodecyl sufate. Higher concentrations of potassium and magnesium ions were required for the translation of albumin mRNA than for total liver mRNAs. These requirements were consistent for the purified albumin as well as when it was a component in the liver mRNA mixture. At the higher potassium or magnesium concentrations, only intact albumin molecules were synthesized, whereas lower concentrations of these ions caused the production of antibody-reactive fragments. These fragments were apparently the result of premature termination of peptide synthesis and not due to endogenous proteolytic activity.


Stimulation of human platelet guanylate cyclase by fatty acids. Guanylate cyclase from human platelets was over 90% soluble, even when assayed in the presence of Triton X-100. A time-dependent increase in activity occurred when the enzyme was incubated at 37 degrees and this spontaneous activation was prevented by dithiothreitol. Arachidonic acid stimulated the soluble enzyme activity approximately 2- to 3-fold. Linear double reciprocal plots of guanylate cyclase activation as a function of arachidonic acid concentration were obtained with a Ka value of 2.1 muM. A Hill coefficient of 0.98 was obtained indicating that one fatty acid binding site is present for each catalytic site. Concentrations of arachidonic acid in excess of 10 muM caused less than maximal stimulation. Dihomo-gamma-linolenic acid and two polyunsaturated 22 carbon fatty acids stimulated the activity of guanylate cyclase to the same degree as did arachidonic acid. The methyl ester of arachidonic acid was much less effective. Diene, monoene, and saturated fatty acids of various carbon chain lengths as well as prostaglandins E1, E2, and F2alpha, had little or no effect. These data indicate that the structural determined required for stimulation by fatty acids of soluble platelet guanylate cyclase is a 1,4,7-octatriene group with its first double bond in the omega6 position. This structural group is similar to the substrate specificity determinants of fatty acid cyclooxygenase, the first enzyme of the prostaglandin synthetase complex. However, conversion of arachidonic acid to a metabolite of the cyclooxygenase pathway did not appear to be required for activation of the cyclase since activation occurred in the 105,000 X g supernatant fraction and pretreatment of this fraction with aspirin did not alter the ability of arachidonic acid to activate guanylate cyclase. Kinetic studies showed that the stimulation of guanylate cyclase by arachidonic acid is primarily an effect on maximal velocity. Arachidonic acid did not alter the concentration of free Mn2+ required for optimal activity. It is concluded that the activity of the soluble form of guanylate cyclase in cell-free preparations of human platelets can be increased by a lipid-protein interaction involving specific polyunsaturated fatty acids.


3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction. Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.


Characterization and kinetics of native and chemically acitvated human liver alcohol dehydrogenases. Acetimidylation of the amino groups of alcohol dehydrogenase from human and horse liver yields several modified enzyme forms, which differ in electrophoretic mobility and can be separated by ion exchange chromatography, but which are similar in kinetic characteristics. The acetimidylated, as well as the methylated, enzymes from human livers of the normal phenotype have increased activity and larger Michaelis and inhibition constants. These results suggest that the human enzyme has amino groups at the active sites, as was shown previously for the horse enzyme. The variant subunit occuring in the enzyme isolated from atypical human livers does not seem to be activated by acetimidylation, which may indicate that substitution of proline for Ala-230 or modifiction of Lys-228 is sufficient to fully activate the enzyme. Results of product inhibition studies of native and modified human enzymes are consistent with an Ordered Bi Bi mechanism. However, the major isoenzyme of native human liver alcohol, dehydrogenase exhibits nonlinear kinetics over a wide range of ethanol concentrations. This result may indicate that subunits with different kinetic characteristics are present or that there is negative cooperativity between subunits. After chemical modification, the kinetic patterns become linear, suggesting that the mechanism is altered.


A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits. A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.


Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase. The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P. These studies led to the scheme below for the ternary complex interconversion. E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations. Step 3 is the hydride transfer. Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values. At lower pH values, the hydride transfer step is also partially rate-determining. The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values. At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step. Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion. Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme. When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred. This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.