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C G Proud

Publications and source records attributed to C G Proud.

115 records · Page 7Linked to original sources
PubMed1984

Metabolite-induced activation of hepatic phosphofructokinase.

Hepatic phosphofructokinase, isolated in a medium containing 100 mM (NH4)2SO4, can be activated by ATP. This metabolite-induced activation was investigated in view of the suggestion that it is related to phosphorylation of phosphofructokinase. The results obtained do not support this interpretation. Inhibitors of protein phosphatases (NaF) and kinases (the Mg++-chelator, ethylene diamine tetraacetic acid) did not affect the recovery of phosphofructokinase. In contrast, media of high ionic strength reduced the phosphofructokinase activity and rendered the enzyme sensitive to ATP-induced activation. Activation was also induced by other known effectors of phosphofructokinase (nucleoside triphosphates, fructose bisphosphates) and was not dependent on Mg++-ions. It is suggested that activation represents ligand-induced reversal of the inactivation of phosphofructokinase which occurs at high ionic strength. The differential sensitivity of phosphofructokinase from fed or starved animals to inactivation and reactivation is discussed.

Adenosine Triphosphate↗
PubMed1982

Regulation of binding of initiator tRNA to eukaryotic initiation factor eIF-2. Effects of the haem-controlled repressor on the kinetics of ternary complex formation.

Ternary complex formation was studied in reticulocyte lysate supernatants and using rat liver eukaryotic initiation factor-2 (eIF-2) preparations. Haem-deficiency reduced the rate of formation of ternary (Met-tRNAf . GTP . eIF-2) complexes by the eIF-2 in reticulocyte supernatants, the reduction being more marked when complex formation was assayed in the absence of GTP-regenerating capacity. Pretreatment with the haem-controlled repressor (HCR) reduced the rate of ternary complex formation by crude (liver) eIF-2. In contrast, complex formation by an almost homogeneous eIF-2 preparation was unaffected by HCR: sensitivity to HCR was however restored by a factor which catalyses exchange of guanine nucleotides bound to eIF-2.

Animals↗
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↗
PubMed1976

The purification and properties of rabbit skeletal muscle glycogen synthase.

Glycogen synthase a was purified over 500-fold by a procedure which involved solubilisation of the enzyme from a protein-glycogen complex by the action of endogenous phosphorylase and debranching enzyme, followed by DEAE-cellulose chromatography, and either gel filtration on Sepharose 4B or fractionation with polyethylene glycol. 15 mg of protein could be obtained from 1000 g of muscle in five days, corresponding to a yield of 20%. The purity was over 90% as judged by gel electrophoresis and ultracentrifugal analysis. The amino acid composition was determined and the absorption coefficient, A1%280 NM, measured refractiometrically was 13.4. Glycogen synthase a sedimented as two major components, both of which were enzymatically active. The smaller species (13.3 S) comprised 85% and the larger species (19.OS) 15% of the material. The molecular weight of the 13.3-S component was determined to be 377000 by high-speed sedimentation equilibrium centrifugation. The subunit molecular weight measured by gel electrophoresis in the presence of sodium dodecylsulphate was 88 000 indicating that the 13.3-S species is a tetramer. The properties of the enzyme are compared to those obtained by other workers.

Amino Acids↗
PubMed1976

The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase.

Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.

Animals↗