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Biomedical subjects

H G Nimmo

Publications and source records attributed to H G Nimmo.

At least 55 records · Page 3Linked to original sources
PubMed1984

Effects of phosphorylation on the kinetic properties of rat liver fructose-1,6-bisphosphatase.

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.

Animals↗
PubMed1984

The allosteric properties of rat liver fructose-1,6-bisphosphatase.

Inhibition of rat liver fructose-1,6-bisphosphatase by AMP was uncompetitive with respect to fructose 1,6-bisphosphate in the absence of fructose 2,6-bisphosphate, but non-competitive in its presence. AMP was unable to bind to the enzyme except in the presence of one of the fructose bisphosphates; the binding stoicheiometry was 2 molecules/tetramer. Increasing concentrations of Mg2+ increased the Hill coefficient h and the apparent Ki for AMP, whereas fructose 2,6-bisphosphate had the opposite effect. Increasing concentrations of both AMP and fructose 2,6-bisphosphate decreased h and increased the apparent Ka for Mg2+. AMP slightly decreased, and Mg2+ slightly increased, the apparent Ki for fructose 2,6-bisphosphate, but each had only small effects on h. These results are interpreted in terms of a new three-state model for the allosteric properties of the enzyme, in which fructose 2,6-bisphosphate can bind both to the catalytic site and to an allosteric site and AMP can bind to the enzyme only when the catalytic site is occupied.

Adenosine Monophosphate↗
PubMed1984

Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML 308.

In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P.M. and Holms, W.H. (1975) J. Gen. Microbiol. 87, 37-51]. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one-dimensional peptide mapping. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were dimers of identical subunits. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.

Acetates↗
PubMed1984

Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308.

Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150. 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. 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. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. 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 a 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. 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.

Amino Acids↗
PubMed1984

The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase.

Isocitrate dehydrogenase kinase can use ATP but not other nucleoside triphosphates as a phosphate donor. It responds hyperbolically to both ATP and isocitrate dehydrogenase. The kinase is inhibited sigmoidally by low concentrations of DL-isocitrate and hyperbolically by ADP, AMP, NADPH, phosphoenolpyruvate and several other effectors. 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. The phosphatase is activated twofold to threefold by AMP, oxaloacetate, pyruvate, phosphoenolpyruvate, 2-oxoglutarate and DL-isocitrate. It is inhibited hyperbolically by NADPH. The pH optima and the Km values for substrates of the kinase and the phosphatase are reported. 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. We suggest that the phosphorylation state of isocitrate dehydrogenase in intact cells is controlled by the levels of isocitrate, phosphoenolpyruvate, NADPH and the adenine nucleotides. This theory can explain many recent observations on the control of the activity of isocitrate dehydrogenase.

Catalysis↗
PubMed1984

A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308.

NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.

Endopeptidases↗
PubMed1983

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase.

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.

Allosteric Regulation↗
PubMed1979

Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase.

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.

Acyltransferases↗
PubMed1978

How does insulin stimulate glycogen synthesis?

One of the important effects of insulin on intracellular metabolism is its ability to stimulate the synthesis of glycogen in muscle and liver. It does this by promoting a net decrease in the extent of phosphorylation of glycogen synthase, the rate-limiting enzyme in the pathway of glycogen synthesis, which increases its activity. Several years ago glycogen synthase was shown to be phosphorylated and inactivated by cyclic AMP-dependent protein kinase in vitro, suggesting that the effect of insulin on glycogen synthesis, and perhaps other intracellular processes, might be explainable in terms of the ability of the hormone to decrease the concentration of tissue cyclic AMP. However, the subsequent failure to detect a decrease in cyclic AMP concentration in muscle under conditions where glycogen synthase activity was stimulated by insulin, coupled with the discovery of a second glycogen synthase kinase whose activity is unaffected by cyclic nucleotides, now suggests the possibility that insulin may regulate the activity of a different class of protein kinase, through its own "second messenger". The identification and characterization of glycogen synthase kinase-2 and recent information about the regulation of glycogen synthase by phosphorylation-dephosphorylation in vitro and in vivo are presented.

Amino Acid Sequence↗
PubMed1977

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

Animals↗
PubMed1976

Protein phosphorylation and hormone action.

Although the scheme hormone leads to raised cyclic AMP levels leads to activated protein kinase leads to phosphorylated protein leads to physiological response may represent an outline for the action of several hormones, in the best understood example, namely regulation of glucogen metabolism in mammalian muscle, the picture is more complex. Modification of phosphorylase kinase by cyclic AMP-dependent protein kinase, after stimulation by adrenaline, leads to phosphorylation of the enzyme at two sites. Activation is associated exclusively with the phosphorylation of the primary site, but the secondary phosphorylation indirectly antagonizes the primary phosphorylation in that it is necessary to render the primary site susceptible to dephosphorylation. The recent separation of two distinct phosphorylase kinase phosphatases specific for the two sites shows that reversal of the hormonal stimulation is controlled by the relative activities of two enzymes with opposing functions. Glycogen synthetase, which is phosphorylated and inactivated by cyclic AMP-dependent protein kinase, is also under the control of insulin. Although insulin appears to stimulate glycogen synthetase by reversal of the inactivation catalysed by the cyclic AMP-dependent protein kinase, tissue cyclic AMP concentrations do not alter. The recent identification of a second glycogen synthetase kinase, unaffected by cyclic AMP, therefore raises the possibility that insulin action may also be mediated through phosphorylation-dephosphorylation mechanisms, which antagonize those mediated through cyclic AMP-dependent protein kinase.

Adenosine Triphosphate↗