Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Intracellular K increases the ouabain-sensitive Na-K exchange in human red blood cells. Pump rate increases hyperbolically with internal K with a K12 for K of 2.58 m-mole/l. red blood cells. Li also stimulates the pump rate, but with a much higher K12. The stimulation does not result from a change in the affinity of the pump for its substrates Na, K or Mg or for the product, phosphate. 2. The effect of cell K on the Na-Na exchange is biphasic. At low concentrations it decreases the exchange rate but then the exchange increases linearly with cell k concentration. 3. Stimulation of the pump rate by internal K can be demonstrated in reconstituted ghosts but only if the ratio of the volume of cells to that of solution at the time of haemolysis is high. Stimulation is not observed if the ghosts contain an efficient system for rephosphorylating ADP to ATP, such as creatine phosphate and creatine kinase, or if the measurements are made with iodoacetamide which inhibits rephosphorylation of ADP by inhibiting the enzyme glyceraldehyde-3-phosphate dehydrogenase. 4. Cells with low internal K and Li have low ATP concentrations, and ATP increases hyperbolically with internal K or Li with the same K12 as does the pump rate. In cells depleted of substrate intracellular K does not stimulate the pump rate. 5. The effect of K and Li on the pump rate probably does not result from enhanced activity of any of the enzymes below phosphofructokinase in the glycolytic pathway.
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PMID:Internal potassium stimulates the sodium-potassium pump by increasing cell ATP concentration. 732 Sep 24

Incorporation of L-[35S]cysteine into rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was detected following incubation of the enzyme in a mixture containing glyceraldehyde-3-phosphate, NAD+ and the labeled cysteine. Insignificant binding occurred in the absence of either the substrate or NAD+, suggesting that formation of an acylated enzyme form was a prerequisite for the binding. Stoichiometry of the binding depended on the number of functioning active centers; up to 4 moles of L-[35S]cysteine bound per mole tetramer with fresh enzyme preparations. The L-[35S]cysteine incorporation depended on pH and was maximal when a group having pKa of 8.5 is protonated. To clarify the relevance of this finding to the effect of SH-containing compounds previously shown to decrease the rate of 3-phosphoglyceroyl-enzyme hydrolysis [Kuzminskaya et al., FEBS Lett. 336 (1993) 208-210], the pH-dependence of the effect of glutathione on the hydrolysis rate was determined and found to be close to the pH-dependence of L-[35S]cysteine binding.
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PMID:Interaction of glyceraldehyde-3-phosphate dehydrogenase with SH-containing compounds: evidence for the binding of L-cysteine and for the dependence of the binding on the functional state of the enzyme. 749 71

Nitric oxide (NO), produced by vascular endothelial cells, mediates both physiological and pathological responses. Although the molecular targets responsible for NO-mediated endothelial cell injury are not known, one candidate is the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study, we investigated the mechanism involved in NO-mediated GAPDH inhibition and found that S-nitrosoglutathione (GSNO) inhibited GAPDH activity in both purified enzyme preparations and endothelial cells. Furthermore, GSNO-mediated GAPDH inhibition occurred by modification of the active site cysteine residue in GAPDH, since increasing concentrations of the substrate, glyceraldehyde-3-phosphate, which interacts with the active site cysteine residue, protected GAPDH from inhibition by GSNO. Although under certain conditions both GSNO and the NO donor, sodium nitroprusside (SNP), led to the covalent NAD(+)-dependent modification of GAPDH, this putative ADP ribosylation was unlikely to be the primary mechanism for inhibition, since the stoichiometry was extremely low, and, in the case of GSNO, inhibition was completely reversed by thiol reagents. Furthermore, GSNO effectively S-nitrosylated GAPDH, and the extent of nitrosylation was linearly correlated with the degree of inhibition such that addition of 1 mole of NO per mole of GAPDH monomer was necessary to inhibit the enzyme. Consistent with this finding, GSNO-mediated GAPDH inhibition was reversible with low-molecular-weight thiols, and the reversal of inhibition correlated with the "denitrosylation" of GAPDH. These results suggest that endothelial GAPDH is a target for NO and that inhibition occurs principally by the reversible S-nitrosylation of the active site cysteine residue in GAPDH.
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PMID:S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation. 757 5

We developed a simple method for the quantitation of triglycerides in electrophoretically separated lipoproteins by specific enzymatic staining. After electrophoresis, glycerol is liberated from triglycerides by the action of cholesterol esterase. Glycerol is oxidized by a sequence of enzymatic reactions. Due to the presence of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in the reaction mixture, two moles of the precipitating dye formazane are generated per mole glycerol. The relative amounts of alpha, pre-beta, and beta lipoproteins are determined by densitometric scanning at 570 nm. Absolute triglyceride concentrations of the respective lipoprotein fractions are calculated from total triglycerides. When tested with purified very low density lipoproteins, the electrophoresis assay was linear between 0.08 and 6.5 g/l pre-beta lipoprotein triglycerides. The intra-assay and inter-assay coefficients of variation were between 5.2% and 9.8%, and between 3.2% and 12.9%, respectively. Comparison of the electrophoresis method with a combined ultracentrifugation/precipitation method in 172 sera resulted in the following correlation coefficients: alpha lipoprotein versus high density lipoprotein triglycerides, r = 0.847; pre-beta lipoprotein versus very low density lipoprotein triglycerides, r = 0.989; beta lipoprotein versus low density lipoprotein triglycerides, r = 0.815. This method is easy to perform, and is a precise and accurate technique for the determination of lipoprotein triglycerides. It is the first reliable method that allows the direct quantification of LDL triglycerides without ultracentrifugation.
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PMID:Determination of triglycerides in lipoproteins separated by agarose gel electrophoresis. 759 4

The ability of glyceraldehyde-3-phosphate dehydrogenase (GAPD) to associate with 3-phosphoglycerate kinase (3-PGK) in human erythrocytes has been studied. It was found that a stable GAPD-3-PGK complex can be isolated from human erythrocyte hemolysates using immobilized monoclonal antibodies that are specific for GAPD. The complex does not dissociate at high ionic strength (up to 0.3 M NaCl) but is decomposed in the presence of specific ligands interacting with GAPD and 3-PGK, e.g., 1,3-diphosphoglycerate. The interaction between GAPD and 3-PGK isolated from human erythrocytes was investigated. To assess the binding parameters, immobilized GAPD and soluble 3-PGK from erythrocytes were used. About 2.3 moles of monomeric 3-PGK (Kd = 2.4 microM) were bound per mole of the immobilized tetramer of GAPD. Under these conditions the rabbit muscle enzymes form more weak (Kd = 3.8 microM), whereas the yeast enzyme--more stable complexes (Kd = 1.5 microM). No such complexes were detected when the enzyme pairs were isolated from phylogenetically distant sources, such as yeast and mammalian tissues. The species specificity of binding of the two enzymes and possible causes of formation of such stable complexes in erythrocyte lysate are discussed.
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PMID:[Glycolytic enzymes in human erythrocytes: association of glyceraldehyde-3-phosphate dehydrogenase with 3-phosphoglycerate kinase]. 807 52

The tetrameric mung bean glyceraldehyde-3-phosphate dehydrogenase is found to bind approximately four moles substrate, glyceraldehyde-3-phosphate, per mole enzyme with Kdiss equal to or less than 9.6 microM at pH 7.3, showing a slight positive cooperativity. Addition of excess substrate to a solution of the enzyme and excess NAD+ leads to a "burst" of NADH formation followed by a slow linear increase (monitored spectrophotometrically). Amount of NADH formed in the burst phase is pH-dependent and is equal to 3.6 moles per mole enzyme at pH 8.6 and above. Presuming four equivalent and independent sites per enzyme molecule (i.e. D2-symmetry), consistent values were obtained for the equilibrium constant of the oxidation-reduction step at different pH and most substrate concentrations. At lower pH (7.3) and high [NAD+]/[substrate] ratios, favouring the C2- symmetry conformation of the enzyme, the magnitude of the burst phase was negligibly small; practically no oxidation reduction reaction took place. Combining these with earlier results on the group transfer step, it is suggested that the oxidation-reduction and group transfer steps of the reaction catalysed by this enzyme require the D2 and C2 symmetry conformations of the enzyme, respectively.
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PMID:Functional significance of protein conformational isomerisation in the glyceraldehyde-3-phosphate dehydrogenase-catalysed reaction. 811 17

Phosphorylation of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) by Ca2+/phospholipid- and Ca2+/calmodulin-dependent protein kinases was shown to take place in rabbit skeletal muscle and brain extracts. The kinases could be "picked up" from the extract, using GPDH immobilized on CNBr-activated Sepharose 4B as an affinity adsorbent. Washing of the column with GPDH solutions resulted in elution of the protein kinases; the same effect was observed when anti-GPDH antibodies were used. The most effective elution took place under the conditions favouring the dissociation of the immobilized GPDH into dimers. Based on these findings, a method for purification of Ca2+/calmodulin-dependent protein kinase has been elaborated, which includes chromatography on phenyl-Sepharose to separate the kinase from GPDH. The susceptibility of GPDH to phosphorylation by tissue protein kinases was confirmed by analyses of GPDH preparations purified from rabbit muscle for endogenous phosphate content: 0.7-1.5 moles of covalently bound phosphate were found per mole of the enzyme.
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PMID:[Phosphorylation of glyceraldehyde-3-phosphate dehydrogenase]. 838 8

Interactions of NAD-dependent dehydrogenases (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, and lactate dehydrogenase, LDH) with band 3 erythrocyte membrane protein and tubulin were characterized. At low ionic strength and un-saturating substrate concentrations, LDH tightly binds to tubulin and is thus inactivated. The Kd of the LDH-tubulin complex was calculated in inhibition and direct binding experiments (15.0 and 13.6 nM, respectively); the stoichiometry of the complex was 1.66 moles of tubulin dimer bound per mole of LDH tetramer. In the presence of 0.15 M NaCl, LDH does not bind to tubulin and tubulin-dependent inhibition of LDH activity is not detected. At low ionic strength, erythrocyte membranes affect both dehydrogenases similarly. GAPDH activity is completely inhibited by excess of erythrocyte membranes (or by excess of cytoplasmic fragment of band 3 protein). Under similar conditions, LDH activity was inhibited by 70% by erythrocyte membranes. In these experiments, 14.8.10(6) GAPDH tetramers or 25.6.10(6) LDH tetramers bound to one erythrocyte ghost (Kd is 0.13 and 0.6 microM, respectively). Increase in ionic strength (0.15 m NaCl) completely abolished the membrane-dependent inhibition of dehydrogenases; however, membranes still bound GAPDH and LDH. Under these conditions, the Kd for GAPDH was increased (up to 4.43 microM), whereas the number of membrane-bound enzyme molecules has not been significantly affected (0.75 nmoles of tetramer per 100 micrograms membrane protein). The Kd for LDH was not changed (0.76 microM), whereas the number of membrane-bound enzyme molecules was decreased (down to 0.48 nmoles of tetramer per 100 micrograms membrane protein). It is suggested that at low ionic strength, the "acidic tails" of band 3 protein and tubulin can interact with positively charged NAD-binding domains of both dehydrogenases thus inhibiting their activity. Increase in ionic strength reduces these interactions, decreasing the binding and inhibition of enzyme activities. At "physiological" ionic strength, catalytically active GAPDH and LDH can possibly bind to various sites of the erythrocyte membrane. This can be important in regulation of the transfer of the common cofactor (NAD/NADH) between their active sites.
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PMID:[Effect of erythrocyte membranes and tubulin on the activity of NAD-dependent dehydrogenases]. 896 25

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a Ki, in the reaction with NADPH at pH 7.05, in the range of 0.2 microM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0. 2 microM). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 microM. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.
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PMID:Effect of NADH-X on cytosolic glycerol-3-phosphate dehydrogenase. 985 31

Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.
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PMID:Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase. 1633 78


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