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Biomedical subjects

A Peterkofsky

Publications and source records attributed to A Peterkofsky.

At least 91 records · Page 5Linked to original sources
PubMed1977

The mechanism of sugar-dependent repression of synthesis of catabolic enzymes in Escherichia coli.

Previous studies have indicated that the Escherichia coli adenylate cyclase (AC) activity is controlled by an interaction with the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS). A model for the regulation of AC involving the phosphorylation state of the PTS is described. Kinectic studies support the concept that the velocity of AC is determined by the opposing contributions of PEP-dependent phosphorylation (V1) and sugar-dependent dephosphorylation (V2) of the PTS proteins according to the expression percent VAC=100/[1 + (Max V2/Max V1)]. Physiological parameters influencing the rate of the PTS are discussed in the framework of their effects on cAMP metabolism. Factors that increase cellular concentration of PEP (and stimulate V1) appear to enhance AC activity while increases in extracellular sugar concentration (which stimulate V2) or internal levels of pyruvate (which inhibit V1) inhibit the activity of this enzyme.

Adenylyl Cyclases↗
PubMed1976

Demonstration of pyroglutamylpeptidase and amidase activities toward thyrotropin-releasing hormone in hamster hypothalamus extracts.

Using a radioimmunoassay method for thyrotropin-releasing hormone, the presence of thyrotropin-releasing hormone-metabolizing activity in various hamster tissues was demonstrated. While there was substantial activity degrading thyrotropin-releasing hormone in hypothalamus, there was a notable absence of such activity in pituitary. The enzymatic activity in the hypothalamus was shown to be soluble and separable into two fractions. Analysis of the metabolic products formed by the two enzymes indicated that one possessed an amidase activity (less than Glu-His-Pro-NH2 leads to less than Glu-His-Pro) and the other possessed pyroglutamylpeptidase activity (less than Glu-His-Pro-NH2 leads to less than Glu+His-Pro-NH2). Other peptides containing NH2-terminal pyroglutamic acid or COOH-terminal amide groups did not block the hydrolysis of thyrotropin-releasing hormone, suggesting that the enzymes were specific. Some inhibitors preferentially blocked the activity of one or the other enzymes. Of possible biological significance is the observation that thyroid-stimulating hormone inhibited the amidase activity while hydrocortisone inhibited the pyroglutamylpeptidase activity.

Amidohydrolases↗
PubMed1976

Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli.

The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues. Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g. alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Analogues with changes in other parts of the molecule (e.g. 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system. This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems. Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively. The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase. In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated. In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated. These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.

Adenylyl Cyclases↗
PubMed1976

Studies on arginyl-tRNA synthetase from Escherichia coli B. Dual role of metals in enzyme catalysis.

Studies carried out in arginyl-tRNA synthetase from Escherichia coli indicate that metals may have two functional roles in the catalytic mechanism. Complete metal activation is observed when MgCl2, MnCl2, CoCl2, or FeCl2 is present at a concentration (5.0 mM) in excess of the total ATP concentration (2.0 mM). When CaCl2 is substituted for MgCl2, activity is not observed unless a small amount (0.1 mM) of MgCl2, MnCl2, CoCl2, FeCl2, or ZnCl2 (unable to produce activity alone at 5.0 mM) is added. A model, based on kinetic data, is proposed in which the enzyme possesses a site for free metal, which, when filled, lowers the Km for all three substrates (arginine, tRNAArg, and metal-ATP) and increases the Vmax of the reaction.

Adenosine Triphosphate↗
PubMed1976

Cyclic nucleotides in bacteria.

The question of the ubiquity of cyclic AMP in bacteria is not yet closed. The recent introduction of more sensitive and reliable assays for cyclic AMP should settle the problem. My prediction is that although there may be some organisms that do not contain cyclic AMP, they probably have some yet undiscovered regulatory nucleotides that play similar roles. Although cyclic AMP has been shown to be unessential for growth of E. coli under optimal laboratory conditions in glucose-containing medium, it undoubtedly can play a role in survival. Cyclic AMP allows bacteria to adapt to a variety of new nutritional conditions. The significance of the observations that cyclic AMP shows a concentration-dependent stimulation or inhibition of growth rate in E. coli is not yet clear. The pathways regulated by cyclic AMP are, for the most part, those which involve carbon metabolism. On the other hand, pathways of nitrogen metabolism are not uniformly regulated by cyclic AMP. In several organisms, some nitrogen pathways are regulated by glutamine synthetase. Specialized processes such as the formation of flagella, fruiting bodies, and buds often appear to be controlled by cyclic AMP. This is similar to the situation in mammalian cells wherein many differentiated functions are regulated by cyclic AMP. Catabolite repression can be explained by an inhibition of the synthesis of cyclic AMP, which does not require an invocation of a primary effect of catabolite action on cyclic AMP phosphodiesterase or on a secretory process, although these two processes are probably of secondary importance. There are some fundamental similarities between the effects of catabolites in inhibiting E. coli adenylyl cyclase and the effects of hormones on mammalian adenylyl cyclase. Both processes require the interaction of the effector with a membrane-bound receptor and may transmit the inhibitory or stimulatory signal to adenylyl cyclase via some coupling factor. Cyclic GMP is clearly present in bacteria, although the features of its molecular biology are just beginning to be laid out. How many other regulatory nucleotides, whether cyclic or linear, remain to be found in bacteria is a problem for the future.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Glucose-sensitive adenylate cyclase in toluene-treated cells of Escherichia coli B.

Toluene treatment of Escherichia coli B makes it possible to measure adenylate cyclase activity directly using [alpha-32-P]-ATP as substrate. In contrast to French press extracts, the activity of adenylate cyclase in toluene-treated cells shows many of the characteristics of the enzyme seen in the intact cell. In both toluene-treated and intact cells the activity of adenylate cyclase is inhibited at least 85% by glucose, while in French press extracts the enzyme activity is much lower and is not sensitive to inhibition by glucose. In toluene-treated cells, glucose inhibits at 10 muM, and the effect is rapid in onset and readily reversible. The activity is not inhibited by glucose 6-phosphate suggesting that glucose is responsible for the inhibition. The measurement of the activity and sensitivity to glucose of adenylate cyclase in toluene-treated cells requires the presence of potassium phosphate in the assay medium. Since it does not increase the activity or sensitivity of the enzyme in the French press extract, it is suggested that potassium phosphate is required for the maintenance of cellular integrity necessary for the activity and sensitivity of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1975

Initiation by methionine of mouse immunoglobulin light chain containing NH-2terminal pyroglutamic acid.

The mechanism of biosynthesis of NH2-terminal pyroglutamic acid has been studied in a mouse plasmacytoma (RPC-20) which produces an immunoglobulin light (lambda) chain containing NH2-terminal pyroglutamic acid. To this end, initation of lambda chain synthesis in plasmacytoma cell suspensions has been investigated. The analysis of radioactive lambda chain synthesis by these cells was accomplished with an antibody preparation specific for the precipitation of lambda chain protein from total plasmacytoma protein. NH2-terminal analysis of plasmacytoma cells labeled with [35S]methionine showed that the ratio of radioactivity in NH2-terminal methionine to total incorporation in lambda chain was greater at 2 min of labeling than at 60 min. However, such a pattern of transient labeling of the NH2 terminus of the lambda chain was not obtained when cells were incubated with tritiated leucine, arginine, or tryptophan. The data indicate that methionine is the initiator amino acid for the synthesis of lambda chain containing NH2-terminal pyroglutamic acid.

Amino Acid Sequence↗
PubMed1975

Interaction of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system with adenylate cyclase of Escherichia coli.

Transient repression by glucose of induced enzyme synthesis involves lowering of intracellular cAMP levels. This glucose effect is partially explained by a glucose inhibition of adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase(cyclizing)]. Since the phosphoenolpyruvate:sugar phosphotransferase system has been implicated in repression phenomena, an investigation was made of adenylate cyclase activity in mutants of that transport system. The results suggest that glucose phosphorylation is not necessary for inhibition of adenylate cyclase since an HPr mutant retained sensitivity to glucose inhibition. The results also suggest that adenylate cyclase activity requires the presence of Enzyme I in a phosphorylated form and that adenylate cyclase activity may be regulated by a phosphorylation-dephosphorylation mechanism.

Adenylyl Cyclases↗
PubMed1975

Formation of chromatographically unique species of transfer ribonucleic acid during amino acid starvation of relaxed-control Escherichia coli.

Examination of the transfer ribonucleic acid (tRNA) produced by starving, relaxed-control (rel minus) strains of Escherichia coli for required amino acids revealed the occurrence of a number of chromatographically unique subspecies. Leucine starvation results in the formation of new isoacceptor species of leucine-, histidine-, arginine-, valine-, and phenylalanine-specific tRNA and quantitative changes in the column profiles of serine, glycine, and isoleucine tRNA. Evidence that the unique tRNA species are synthesized de novo during amino acid starvation comes from the findings that the major unique leucine isoacceptor species is not formed in stringent control cells or in rel minus cells starved for uracil or treated with rifampin. Furthermore, heat treatment of the unique leucine tRNA does not alter its chromatographic behavior, indicating that the species is not an aggregate or nuclease-damaged form of a normal isoacceptor tRNA. The methyl acceptor activities of tRNA from leucine-starved and nonstarved rel+ or rel minus cells were found to be essentially the same. This result and the finding that the chromatographic behavior of the unique leucine-specific tRNA was not altered after treatment with tRNA methylase suggests that gross methyl deficiency is probably not the biochemical basis for the occurrence of the unique species.

Acylation↗
PubMed1974

Glucose inhibition of adenylate cyclase in intact cells of Escherichia coli B.

Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since alpha-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium.

Adenylyl Cyclase Inhibitors↗
PubMed1973

Measurements of rates of adenosine 3':5'-cyclic monophosphate synthesis in intact Escherichia coli B.

A method for labeling adenosine 3':5'-cyclic monophosphate in vivo from precursor adenosine followed by quantitative analysis of the labeled nucleotide is described. When the labeling period is short and the specific activity of the ATP pool is determined, the rate of incorporation of radioactivity corresponds to a determination of adenylate cyclase activity in vivo. In E. coli B the specific activity of adenylate cyclase in vivo varies under different growth conditions. Under all conditions tested, the adenylate cyclase activity in vivo is great enough to account for the pattern of accumulation of cyclic AMP. This is in contrast to previous adenylate cyclase assays in vitro, where the measured enzyme activities were insufficient to account for the amounts of cyclic AMP accumulated.

Adenosine↗