Evidence for absence of an interaction between purified 3-phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase

The possibility of a functional complex formation between glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 3-phosphoglycerate kinase (EC. 2.7.2.3), enzymes catalysing two consecutive reactions in glycolysis has been investigated. Kinetic analysis of the coupled enzymatic reaction did not reveal any kinetic sign of the assumed interaction up to 4 X 10(-6) M kinase and 10(-4) M dehydrogenase. Fluorescence anisotrophy of 10(-7) M or 2 X 10(-5) M glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocynate did not change in the presence of non-labeled 3-phosphoglycerate kinase (up to 4 X 10(-5) M). The frontal gel chromatographic analysis of a mixture of the two enzymes (10(-4) M dehydrogenase) could not reveal any molecular species with the kinase activity having a molecular weight higher than that of 3-phosphoglycerate kinase. Both types of physicochemical measurements were also performed in the presence of substrates of the kinase and gave the same results. The data seem to invalidate the hypothesis that there is a complex between purified pig muscle glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Hexokinase-coupled radioassays: Glyceraldehyde-3-phosphate dehydrogenase and enolase

A general method for the assay of enzymes which produce ATP, or are susceptible to be coupled to ATP-producing enzymes, is described. We have applied it to the assay of glyceraldehyde-3-phosphate dehydrogenase and enolase. For these enzymes, the product of the reaction, 1,3-bisphosphoglycerate or phosphoenolpyruvate, were coupled to ADP and either phosphoglycerate kinase or pyruvate kinase, respectively. The ATP formed in both cases is used by hexokinase plus labeled glucose to produce labeled glucose 6-phosphate which is quantitatively separated in small Dowex 1 columns and measured by liquid scintillation spectrometry. The conditions described permitted the detection of 0.1 mU of glyceraldehyde-3-phosphate dehydrogenase or enolase. As a further example of the sensitivity of the radioassay, effluents of a CM-cellulose column charged with an extract prepared from one single frog oocyte were analyzed and shown to contain a single enolase and two glyceraldehyde-3-phosphate dehydrogenase fractions.     

Immobilized glyceraldehyde-3-phosphate dehydrogenase forms a complex with phosphoglycerate kinase

Yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) covalently attached to CNBr-activated Sepharose 4B was shown to be capable of binding soluble yeast phosphoglycerate kinase (PGK) in the course of incubation in the presence of an excess of 1,3-diphosphoglycerate. The association of the matrix-bound and soluble enzymes also occurred if the kinase was added to a reaction mixture in which the immobilized glyceraldehyde-3-phosphate dehydrogenase, NAD, glyceraldehyde-3-phosphate and Pi had been preincubated. Three kinase molecules were bound per a tetramer of the immobilized dehydrogenase and one molecule per a dimer. An immobilized monomer of glyceraldehyde-3-phosphate dehydrogenase was incapable of binding phosphoglycerate kinase. The matrix-bound bienzyme complexes were stable enough to survive extensive washings with a buffer and could be used repeatedly for activity determinations. Experimental evidence is presented to support the conclusion that 1,3-diphosphoglycerate produced by the kinase bound in a complex can dissociate into solution and be utilized by the dehydrogenase free of phosphoglycerate kinase.     

Properties of two high-molecular-mass forms of glyceraldehyde-3-phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity.

Two high-Mr forms of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach leaf can be separated by DEAE-cellulose chromatography. One form, the high-Mr glyceraldehyde-3-phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G.R., Ortwerth, B.J. & Koeppe, O.J. (1970) J. Biol. Chem. 245, 4193-4198]. The other, a glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol. This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high-Mr glyceraldehyde-3-phosphate dehydrogenase, but also of a third subunit, R (40.5 kDa) comigrating with that from the active phosphoribulokinase of spinach. Incubation of the complex with dithiothreitol markedly stimulated both its phosphoribulokinase and NADPH-dependent dehydrogenase activities. This dithiothreitol-induced activation was accompanied by depolymerisation to give two predominantly NADPH-linked tetrameric glyceraldehyde-3-phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high-Mr glyceraldehyde-3-phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2). Possible structures suggested for the glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.     

Mechanism of 1,3-bisphosphoglycerate transfer from phosphoglycerate kinase to glyceraldehyde-3-phosphate dehydrogenase

1. The kinetics of 1,3-bisphosphoglycerate binding to glyceraldehyde-3-phosphate dehydrogenase have been examined by stopped-flow techniques in the absence and presence of phosphoglycerate kinase, using enzyme concentrations in the range 0.5-40 microM. Rate and equilibrium constant estimates for the interaction of the ligand with the two enzymes are reported. 2. The kinetics of ligand transfer from the binary complex of bisphosphoglycerate and phosphoglycerate kinase to the binary complex of NAD+ and glyceraldehyde-3-phosphate dehydrogenase conform excellently to the predictions of a standard free-diffusion mechanism and exhibit no detectable contributions from a mechanism of direct (channelized) transfer of bisphosphoglycerate between the two enzymes. 3. Previously reported evidence that the binary complex of bisphosphoglycerate and phosphoglycerate kinase may act (in the presence of NADH) as a substrate for glyceraldehyde-3-phosphate dehydrogenase according to Michaelis-Menten kinetics is based on a misinterpretation of the experimental observations that can be attributed to neglect of the autocatalytic effect of NAD+ produced during the reaction. Experiments performed under conditions where the autocatalytic effect of NAD+ is eliminated provide clear evidence that the kinetics of utilization of the kinase-bisphosphoglycerate complex for enzymic NADH reduction are consistent with prior dissociation of the complex according to a free-diffusion mechanism of metabolite transfer and incompatible with a mechanism of direct metabolite transfer. 4. A kinetic argument is presented which renders implausible the very idea that direct metabolite transfer between 'soluble' consecutive enzymes in metabolic pathways may offer any catalytic advantages in comparison to metabolite transfer by free diffusion. A mechanism of direct metabolite transfer seems intuitively attractive only because one tends to disregard the diffusional processes required to bring the consecutive enzymes together and to separate them when the transfer has been completed. Direct metabolite transfer would be expected to be catalytically advantageous only in tightly bound multienzyme complexes showing no kinetically significant tendency to dissociate. 5. It is concluded that mechanisms of direct metabolite transfer have not been convincingly demonstrated to apply, nor are they likely to apply, between 'soluble' consecutive enzymes in metabolic pathways, at least not in the glycolytic sequence of reactions.     

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.     

Characterization of a nitric-oxide-catalysed ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.

Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of auto-[32P]ADP-ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP-ribose-cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD-glycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADP-ribosylations were carried out at alkaline pH values using 10 microM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD+ and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction.     

Changing kinetic properties of the two-enzyme phosphoglycerate kinase/NADP-linked glyceraldehyde-3-phosphate dehydrogenase couple from pea chloroplasts during photosynthetic induction

The kinetics of the two enzyme phosphoglycerate kinase (EC 2.7.2.3)/NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) couple are negatively cooperative and will also fit a model for two enzymes acting on one substrate. When the chloroplast is illuminated apparent negative cooperativity is reduced; maximal velocity of only one of the two enzymes in the two-enzyme model is increased. Even after light activation the activity of glyceraldehyde-3-phosphate dehydrogenase appears to be too low to support photosynthesis at calculated levels of glycerate-1,3-bisphosphate in isolated chloroplasts (Marques, I.A., Ford, D.M., Muschinek, G. and Anderson, L.E. (1987) Arch. Biochem. Biophys. 252, 458–466). The activity of the coupled reaction is apparently sufficient to support observed rates of CO₂ fixation, which suggests that glycerate-1,3-bisphosphate may be channeled from the kinase to the dehydrogenase in vivo.     

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes

Utilizing yeast strains containing insertion mutations in each of the three glyceraldehyde-3-phosphate dehydrogenase structural genes, the level of expression of each gene was determined in logarithmically growing cells. The contribution of the TDH1, TDH2, and TDH3 gene products to the total glyceraldehyde-3-phosphate dehydrogenase activity in wild type cells is 10-15, 25-30, and 50-60%, respectively. The relative proportions of expression of each gene is the same in cells grown in the presence of glucose or ethanol as carbon source although the total glyceraldehyde-3-phosphate dehydrogenase activity in cells grown in the presence of glucose is 2-fold higher than in cells grown on ethanol. The polypeptides encoded by each of the structural genes were identified by two-dimensional polyacrylamide gel electrophoresis. The TDH3 structural gene encodes two resolvable forms of glyceraldehyde-3-phosphate dehydrogenase which differ by their net charge. The apparent specific activity of glyceraldehyde-3-phosphate dehydrogenase encoded by the TDH3 structural gene is severalfold lower than the enzymes encoded by TDH1 or TDH2. The polypeptides encoded by the TDH2 or TDH3 structural genes form catalytically active homotetramers. The apparent Vmax for the homotetramer encoded by TDH3 is 2-3-fold lower than the homotetramer encoded by TDH2. Evidence is presented that isozymes of glyceraldehyde-3-phosphate dehydrogenase exist in yeast cells, however, the number of different isozymes formed was not established. These data confirm that the three yeast glyceraldehyde-3-phosphate dehydrogenase genes encode catalytically active enzyme and that the genes are expressed at different levels during logarithmic cell growth.     

Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase. Difference in the mode of modification by pyrene maleimide

Pyrene maleimide is shown to be a 'half of the sites' reagent for glutamate dehydrogenase and for glyceraldehyde-3-phosphate dehydrogenase. The modified residues are identified as cysteine-115 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase. The two enzymes react differently with pyrene maleimide. Whereas the hydrophobic environment of cysteine-115 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase. Glutamate dehydrogenase activity is unaltered by the modification: glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.     

Immobilized hybrids of glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) immobilized on CNBr-activated Sepharose 4-B has been subjected to dissociation to obtain matrix-bound dimeric species of the enzyme. Hybridization was then performed using soluble glyceraldehyde-3-phosphate dehydrogenase isolated from rat skeletal muscle. Immobilized hybrid tetramers thus obtained were demonstrated to exhibit two distinct pH-optima of activity characteristic of the yeast and muscle enzymes, respectively. The results indicate that under appropriate conditions the activity of each of the dimers composing the immobilized hybrid tetramer can be studied separately.     

Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560,000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42,000 conferring phosphoribulokinase activity and G a subunit of 39,000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42,000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39,000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography. Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase. Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

Identification of the 37-kDa protein displaying a variable interaction with the erythroid cell membrane as glyceraldehyde-3-phosphate dehydrogenase

In previous studies from this laboratory we isolated and characterized a 37-kDa protein that was associated with the membrane of erythroid cells. The polypeptide appeared to undergo a lineage-specific alteration in its interaction with the membrane during erythroid development and migrated as a family of isoelectric focusing variants during analyses on two-dimensional gels. We report here that the 37-kDa protein is homologous to the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). This conclusion was reached from the results of several experimental approaches comparing the biochemical and genetic properties of the 37-kDa protein (p37) with authentic glyceraldehyde-3-phosphate dehydrogenase. Peptide maps of highly purified p37 and glyceraldehyde-3-phosphate dehydrogenase, generated with Staphylococcus V8 protease, were identical. The nucleotide sequence of a cDNA clone encoding p37 was nearly identical to the published sequence for genes encoding glyceraldehyde-3-phosphate dehydrogenase. These results suggest that the interaction of the enzyme with the red cell membrane is more complex than previously envisioned. The existence of subpopulations of glyceraldehyde-3-phosphate dehydrogenase molecules is envisioned that exhibit different levels of enzyme activity and bind to the red cell membrane with varying affinities.     

Antivibrionic activity of some glycolytic cycle enzymes of animal cells.

The antivibrionic activity of crystalline preparations of five enzymes of the glycolytic cycle of animals cells was investigated. Phosphorylase "a" (0.5 mg/ml), aldolase (15 mg/ml) and pyruvate kinase (0.1 mg/ml) were found to inhibit the proliferation of Vibrio cholerae cells; phosphoglucomutase and glyceraldehyde-3-phosphate dehydrogenase at a concentration of 0.25 mg/ml were found to be vibriocidal. A mixture of these enzymes containing 0.062 mg/ml of phosphorylase "a" and 0.125 mg/ml of each phosphoglucomutase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase showed vibriocidal activity.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.