Inhibition of phosphoglucomutase activity by lithium alters cellular calcium homeostasis and signaling in Saccharomyces cerevisiae.
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Phosphoglucomutase is a key enzyme of glucose metabolism that interconverts glucose-1-phosphate and glucose-6-phosphate. Loss of the major isoform of phosphoglucomutase in Saccharomyces cerevisiae results in a significant increase in the cellular glucose-1-phosphate-to-glucose-6-phosphate ratio when cells are grown in medium containing galactose as carbon source. This imbalance in glucose metabolites was recently shown to also cause a six- to ninefold increase in cellular Ca2+ accumulation. We found that Li+ inhibition of phosphoglucomutase causes a similar elevation of total cellular Ca2+ and an increase in 45Ca2+ uptake in a wild-type yeast strain grown in medium containing galactose, but not glucose, as sole carbon source. Li+ treatment also reduced the transient elevation of cytosolic Ca2+ response that is triggered by exposure to external CaCl2 or by the addition of galactose to yeast cells starved of a carbon source. Finally, we found that the Ca2+ over-accumulation induced by Li+ exposure was significantly reduced in a strain lacking the vacuolar Ca2+-ATPase Pmc1p. These observations suggest that Li+ inhibition of phosphoglucomutase results in an increased glucose-1-phosphate-to-glucose-6-phosphate ratio, which results in an accelerated rate of vacuolar Ca2+ uptake via the Ca2+-ATPase Pmc1p. (+info)
Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors.
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Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme. (+info)
Properties of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.
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The properties of three phosphatases from Salmonella typhimurium have been examined. A cyclic 2',3'-nucleotide phosphodiesterase (EC 3.1.4.d) hydrolyzes cyclic 2',3'-purine and -pyrimidine nucleotides, as well as 3'-mononucleotides, and has a pH optimum of about 7.5. It requires divalent cations for activity and has a molecular weight of 67,000. Acid hexose phosphatase (EC 3.1.2.2) possesses activity towards hexose phosphates as well as other sugar phosphates. The enzyme is apparently a dimer of 37,000-dalton subunits. Nonspecific acid phosphatase (EC 3.1.3.2) hydrolyzes a variety of phosphate esters, including nucleotides and sugar phosphates. The enzyme also hydrolyzes the phosphoric anhydride bonds of pyrophosphate and nucleotides. Michaelis constants of the nonspecific acid phosphatase for several of its substrates are in the 1 to 2 mM range. Nonspecific acid phosphatase is a dimer of 27,000-dalton subunits. (+info)
CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation.
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The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate. (+info)
Effects of insulin-like growth factor I on the rates of glucose transport and utilization in rat skeletal muscle in vitro.
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1. The effects of insulin-like growth factor I (IGF-I) on the rates of glucose transport and utilization and its interaction with insulin were investigated in rat soleus muscle in vitro. IGF-I increased the rates of glucose transport, lactate formation, glycogen synthesis and the flux of glucose to hexose monophosphate, but it had no effect on the rate of glucose oxidation or glycogenolysis. 2. In the absence of insulin, low levels of IGF-I (0-30 ng/ml) increased the rate of glycolysis and the content of fructose 2,6-bisphosphate, but the content of glucose 6-phosphate remained unaltered; at higher levels of IGF-I (300-3000 ng/ml) the rate of glycolysis and the content of fructose 2,6-bisphosphate showed a further modest increase, but the content of glucose 6-phosphate doubled. Similar changes were seen when the level of insulin was increased from basal (0-0.4 ng/ml) to maximal (40 ng/ml). 3. Neither IGF-I nor insulin affected the contents of ATP, ADP, AMP, phosphocreatine or citrate. 4. Maximal concentrations of IGF-I increased the rate of lactate formation to a greater extent than did maximal concentrations of insulin. 5. In the presence of IGF-I, the rate of glucose utilization was less responsive to insulin. 6. The results suggest that, in rat skeletal muscle: (a) IGF-I increases the rates of glucose transport and utilization independently of insulin, and has a preferential effect on the rate of lactate formation; (b) the effects of IGF-I and insulin are not additive; (c) in addition to its effects on glucose transport, IGF-I increases the rate of glycogen synthesis and may stimulate glycolysis at the level of 6-phosphofructokinase; (d) changes in the content of fructose 2,6-bisphosphate may be part of the mechanism to regulate glycolytic flux in skeletal muscle in response to either IGF-I or insulin. (+info)
Pyridoxal phosphate-dependent conformational states of glycogen phosphorylase as probed by interconverting enzymes.
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The interaction between pyridoxal 5'-phosphate and the convertible serine of glycogen phosphorylase has been investigated by using: specific interconverting enzymes, phosphorylase kinase and phosphorylase phosphatase; effectors, glucose and glucose 6-phosphate; and a protein kinase and trypsin. Both phosphorylase kinase and phosphorylase phosphatase utilized the native protein while having little influence on the apoprotein. Removal of a peptide containing the critical serine residue gave phosphorylase b' from which the pyridoxal 5'-phosphate in phosphorylase has an important effect on enzymic interconversion. (+info)
Hypoxia promotes relaxation of bovine coronary arteries through lowering cytosolic NADPH.
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Hypoxia relaxes endothelium-denuded bovine coronary arteries (BCA) through mechanisms that do not appear to involve reactive oxygen species, prostaglandins, or nitric oxide. Because of similarities in the relaxation of BCA to hypoxia (Po(2) = 8-10 Torr) and inhibitors of the pentose phosphate pathway (PPP) including 6-aminonicotinamide and epiandrosterone, we measured NADPH and NADP and found that hypoxia caused NADPH oxidation (decreased NADPH/NADP). The relaxation to hypoxia was similar to previously reported properties of relaxation to PPP inhibitors in that both responses were associated with glutathione oxidation and depressed intracellular calcium release and calcium influx-mediated contractile responses. Inhibitors of potassium channels had minimal effects on these relaxation responses. Relaxation to hypoxia and PPP inhibitors were attenuated by a thiol reductant (3 mM dithiothreitol) and by eliciting contraction with an activator of protein kinase C (phorbol 12,13-dibutyrate). In the presence of contraction to U-46619, relaxation to hypoxia and PPP inhibitors were attenuated by the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump inhibitor 200 microM cyclopiazonic acid and by 10 mM pyruvate. Hypoxia decreased BCA levels of glucose-6-phosphate but not ATP. Pyruvate prevented the hypoxia-elicited decrease in glucose-6-phosphate and glutathione oxidation, and it increased NADPH levels under hypoxia to levels observed under normoxia. Thus hypoxia causes a metabolic stress on the PPP that promotes BCA relaxation through processes controlled by lowering the levels of cytosolic NADPH. (+info)
A two-stage one-pot enzymatic synthesis of TDP-L-mycarose from thymidine and glucose-1-phosphate.
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This report describes a procedure combining six enzymes native to Escherichia coli or Salmonella typhi, such as thymidine kinase (TK), thymidylate kinase (TMK), nucleoside diphosphate kinase (NDK), pyruvate kinase (PK; for ATP regeneration), TDP-glucose synthetase (RfbA), and TDP-glucose 4,6-dehydratase (RfbB), with five enzymes from Streptomyces fradiae, such as TylX3, TylC1, TylC3, TylK, and TylC2, that resulted in the biosynthesis of TDP-l-mycarose from glucose-1-phosphate and thymidine. This two-stage one-pot approach can be readily applied to the synthesis of other unusual sugars. (+info)