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1. Isocitrate dehydrogenase kinase can use ATP but not other nuclcoside triphosphates as a phosphate donor. It responds hyperbolically to both ATP and isocitrate dehydrogenase. 2. The kinase is inhibited sigmoidally by low concentrations of DL-isocitrate and hyperbolically by ADP, AMP, NADPH, phosphoenolpyruvate and several other effectors. 3. Isocitrate dehydrogenase phosphatase requires a nucleotide for activity; ADP and ATP are the best activators. The phosphatase responds hyperbolically to ADP or ATP, to Mg2+ ions and to phosphorylated isocitrate dehydrogenase. 4. The phosphatase is activated twofold to threefold by AMP, oxaloacetate, pyruvate, phosphoenolpyruvate, 2-oxoglutarate and DL-isocitrate. It is inhibited hyperbolically by NADPH. 5. The pH optima and the Km values for substrates of the kinase and the phosphatase are reported. 6. We propose that the role of the phosphorylation of isocitrate dehydrogenase during growth of Escherichia coli on acetate is to render this enzyme rate-limiting in the citric acid cycle; this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass. 7. We suggest that the phosphorylation state of isocitrate dehydrogenase in intact cells is controlled by the levels of isocitrate, phosphoenolpyruvate, NADPH and the adenine nuclcotides. This theory can explain many rccent observations on the control of the activity of isocitrate dehydrogenase. [ABSTRACT FROM AUTHOR]

1. Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by a procedure involving fractionation with (NH4)2SO4 and chromatography on DEAEcellulose, blue-dextran-Sepharose and Sephadex G150. 2. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D. C. and Koshland, D. E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. 3. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. 4. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. 5. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was n good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. 6. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity. [ABSTRACT FROM AUTHOR]

Reports on kinetic comparisons between soluble and membrane-bound glutaminase preparations from pig brain. Dependence of activities on pH and in their inhibition by borate and some glutamine analogs; Responses to phosphate and their inhibition by glutamate; Sigmoid dependence of initial velocity on glutamine concentration; Inhibition by ammonium ions; Catalysis of the hydrolysis of L-glutamine by the enzyme glutaminase to form L-glutamate plus ammonium ions.

A simple and convenient procedure is described for the isolation in good yield of two aminotransferases from various strains of Escherichia coli. On the basis of their substrate specificities one of the enzymes has been classified as an aromatic amino acid aminotransferase and the other as an aspartate aminotransferase, but both act on a wide range of substrates. Pyridoxal phosphate is bound more strongly to the aspartate aminotransferase than to the aromatic amino transferase which cannot be fully re-activated after removal of the prosthetic group. Both enzymes are composed of two subunits which appear to be identical. [ABSTRACT FROM AUTHOR]

One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here, we investigated the role of RNA-binding splicing factors (SFs) in temperature-sensitive AS of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterized, in wild type plants, temperature-associated isoform switching and expression patterns for SF transcripts from a high-resolution temperature and time series RNA-seq experiment. In addition, we employed quantitative RT-PCR of SF mutant plants to explore the role of the SFs in cooling-associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of the 5'UTR of LHY. Moreover, the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5'UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2 °C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF-mediated coupling of the perception of temperature to post-transcriptional regulation of the clock., (© 2018 The Authors. Plant, Cell & Environment Published by John Wiley & Sons Ltd.)