Interaction between glyceraldehyde-3-phosphate-dehydrogenase and lactate dehydrogenase

Physical interaction between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase was detected by means of matrix immobilization technique. Glyceraldehyde-3-phosphate dehydrogenase covalently bound to CNBr-activated Sepharose 4B was capable of forming a complex with soluble lactate dehydrogenase with a stoichiometry of 0.8 mole of lactate dehydrogenase per mole of glyceraldehyde-3-phosphate dehydrogenase and KD of 0.385 microM at pH 6.5. The bienzyme association weakened when pH changed to 7.0 (the KD increased to 1.25 microM).     

Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase and its functional consequences.

E. coli D-glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli 3-phosphoglycerate kinase with a stoichiometry of 1.77 +/- 0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kd of 1.03 +/- 0.68 microM (10 mM sodium phosphate, 0.15 M NaCl). No interaction was detected between E. coli D-glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle 3-phosphoglycerate kinase. The species-specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E. coli D-glyceraldehyde-3-phosphate dehydrogenase and E. coli 3-phosphoglycerate kinase, which consists of an increase in steady-state rate of the coupled reaction.     

Binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules

The interaction of glyceraldehyde 3-phosphate dehydrogenase with microtubules has been studied by measurement of the amount of enzyme which co-assembles with in vitro reconstituted microtubules. The binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules is a saturable process; the maximum binding capacity is about 0.1 mole of enzyme bound per mole of assembled tubulin. Half saturation of microtubule binding sites is obtained at a concentration of glyceraldehyde 3-phosphate dehydrogenase of about 0.5 µM Glyceraldehyde 3-phosphate dehydrogenase (between 0.1 and 2 µM) induces a concentration-dependent increase a) in the turbidity of the microtubule suspension without alteration of the net amount of polymer formed and b) in the amount of microtubule protein polymers after cold microtubule disassembly. There is a linear relationship between the intensity of the glyceraldehyde 3-phosphate dehydrogenase-induced effects and the amount of microtubule-bound enzyme. The specificity of the association of glyceraldehyde 3-phosphate dehydrogenase to microtubules has been documented by copolymerization experiments. Assembly-disassembly cycles of purified microtubules in the presence of a crude liver soluble fraction results in the selective extraction of a protein with an apparent molecular weight of 35 000 identified as the monomer of glyceraldehyde 3-phosphate dehydrogenase by peptide mapping and immunoblotting.In conclusion, microtubules possess a limited number of binding sites for glyceraldehyde 3-phosphate dehydrogenase. The binding of the glycolytic enzyme to microtubules shows a considerable specificity and is associated with alterations of assembly and disassembly characteristics of microtubules.Abbreviations Mes 2(N-morpholinoethane) sulfonic acid - EGTA ethylene glycol bis (-aminoethyl-ester)N,N,N,N tetraacetic acid - EDTA thylene diamine tetraacetic acid     

Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by fluorescein-5'-isothiocyanate: evidence that the dye participates in the interaction

An interaction of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled with FITC was studied by following the changes in fluorescence intensity of the bound dye. The association between the two enzymes was found to be a rather slow process characterized by a second order rate constant of 1.1 +/- 0.2.10(3) M-1 s-1, the KD of the complex between apoenzymes being 3.2.10(-7) M. The stability of the complex increased upon increase of temperature and ionic strength of the medium, suggesting a hydrophobic character of association. The ligands which bind at the active centers of the two enzymes (NAD+, ATP, 3-phosphoglycerate) weakened the bienzyme association. Unlabeled 3-phosphoglycerate kinase was unable to displace the FITC-labeled enzyme from the complex. Taken together, the results indicate that interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by FITC is assisted by the dye, which may bind at nucleotide-binding sites of GPDH. No interaction was observed between the FITC-labeled 3-phosphoglycerate kinase and lactate dehydrogenase, which suggests that protein-protein interaction at specific "recognition" sites may be a prerequisite for the complex formation.     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

Parallel evolution of pairs of dehydrogenase isoenzymes

Summary Lactate dehydrogenase and glycerol 3-phosphate dehydrogenase are metabolically coupled by the anaerobic dismutation of glyceraldehyde 3-phosphate and by the NAD redox state. This causes the concentrations of lactate and glycerol 3-phosphate to accumulate proportionally during anaerobic muscle contraction; these concentrations are high relative to those in aerobic tissues such as liver. We show that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken breast muscle haveKm values for lactate and glycerol 3-phosphate, respectively, that are 10-fold higher than theKm values measured for the lactate dehydrogenase and glycerol 3-phosphate dehydrogenase isoenzymes from chicken liver. The association of proportionally higherKm values with the potential for proportionally higher accumulation of substrates suggests that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken muscle have evolved in parallel as a coupled metabolic unit distinct from the coupled isoenzymes in liver. The parallelism observed for the reduced substrates extends to the oxidized substrates, and to the coenzymes, NAD⁺ and NADH.     

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.     

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.     

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.     

Triiodothyronine depresses the NAD-linked glycerol-3-phosphate dehydrogenase activity of cultured neonatal rat heart cells

The activity of NAD-linked alpha-glycerol-3-phosphate dehydrogenase (NAD-G3PDH; EC 1.1.1.8) was depressed by 35% when the thyroid hormone 3,3',5-triiodo-L-thyronine (20 micrograms/liter) was added to the serum-free, hormonally supplemented medium of cultured neonatal rat heart cells. The degree of depression was greater (65%) when the medium contained normal serum levels of hydrocortisone and insulin. There is a dramatic inverse dose-response relationship between triiodothyronine levels and NAD-G3PDH activity. The classic elevation by thyroid hormones of the FAD-linked alpha-glycerol-3-phosphate dehydrogenase (FAD-G3PD; EC 1.1.99.5) was observed concurrently. The medium-glucose depletion rate in triiodothyronine-free cells was depressed 32% through 11 days-in-culture, indicating reduced glycolytic activity. The activities of nine other metabolically important enzymes which were measured during this study, including hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, malate dehydrogenase, NAD-isocitrate dehydrogenase, NADH cytochrome c reductase, and succinic cytochrome c reductase, did not respond to varying triiodothyronine concentrations.     

The rate constants of the reaction of hydroxyl radicals (*OH) with alcohol dehydrogenase and Glyceraldehyde-3-phosphate dehydrogenase

The rate constants of the reactions of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase with hydroxyl radicals were determined using the method of steady-state competitive reactions. Ethanol was used as a scavenger of hydroxyl radicals. The rate constants of the reactions of hydroxyl radicals with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were found to be 2.8 x 10(12) dm(3) mol(-1) s(-1), and 1.6 x 10(12) dm(3) mol(-1) s(-1), respectively.     

The D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase complex from rabbit skeletal muscles

The experimental conditions favouring the association of Sepharose-bound D-glyceraldehyde-3-phosphate dehydrogenase with soluble 3-phosphoglycerate kinase were studied. Acylation of D-glyceraldehyde-3-phosphate dehydrogenase by 1.3-bisphosphoglycerate was found to be a prerequisite for the complex formation.     

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.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Identification of the arylazido-beta-alanyl-NAD+-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry

We have identified the site labeled by arylazido-beta-alanyl-NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+) in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry. This NAD+ photoaffinity analogue has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in a very specific manner and probably at the active site of the enzyme [Chen, S., Davis, H., Vierra, J. R., & Guillory, R. J. (1984) Biochem. Biophys. Stud. Proteins Nucleic Acids, Proc. Int. Symp., 3rd, 407-425]. The label is associated exclusively with a tryptic peptide that has the sequence Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue at position seven was predominantly labeled and suggested to be the site modified by arylazido-beta-alanyl-NAD+. This cysteine residue corresponds to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity. The present investigation clearly demonstrates that arylazido-beta-alanyl-NAD+ is a useful photoaffinity probe to characterize the active sites of NAD(H)-dependent enzymes.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Thermodynamic analysis of the emergence of new regulatory properties in a phosphoribulokinase-glyceraldehyde 3-phosphate dehydrogenase complex

Glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase exist as stable enzymes and as part of a complex in Chlamydomonas reinhardtii. We show here that phosphoribulokinase exerts an imprinting on glyceraldehyde 3-phosphate dehydrogenase, which affects its catalysis by decreasing the energy barrier of the reactions with NADH or NADPH by 3.8 +/- 0.5 and 1.3 +/- 0.3 kJ.mol(-1). Phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase within the complex are regulated by NADP(H) but not by NAD(H). The activities of the metastable phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase released from the complex preincubated with NADP(H) are different from those of the metastable enzymes released from the untreated complex. NADP(H) increases phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase activities with a (~)K(0.5 (NADP)) of 0.68 +/- 0.16 mm and a (~)K(0.5 (NADPH)) of 2.93 +/- 0.87 mm and decreases NADH-dependent activity. 1 mm NADP increases the energy barrier of the NADH-glyceraldehyde 3-phosphate dehydrogenase-dependent reaction by 1.8 +/- 0.2 kJ.mol(-1) and decreases that of the reactions catalyzed by phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase by 3 +/- 0.2 and 1.2 +/- 0.3 kJ.mol(-1), respectively. These cofactors have no effect on the independent stable enzymes. Therefore, protein-protein interactions may give rise to new regulatory properties.     

Crystallization and preliminary crystallographic analysis at low resolution of the allosteric l-lactate dehydrogenase from Lactobacillus casei

The allosteric l-lactate dehydrogenase from Lactobacillus casei has been crystallized in its complex with the activators fructose-1,6-diphosphate and Co²⁺. The enzyme crystallizes in space group C2 with six tetramers in the unit cell. At very low resolution, 00l reflexions are absent for l ≠ 3n. The orientation of the molecular axes has been determined using the rotation function. All tetramers in the unit cell exhibit excellent 222 symmetry, and the overall arrangement resembles the packing that would be expected in the higher symmetry space group P3₁21. Comparison with the apo-enzyme structure of M4-lactate dehydrogenase from dogfish indicates high structural similarity between these enzymes and allowed us to identify the molecular axes of L. caseil-lactate dehydrogenase in terms of the “standard” molecular co-ordinate system P, Q, R. The similarity of both enzymes is good enough to allow the structure determination of L. caseil-lactate dehydrogenase by molecular replacement using the dogfish enzyme as a model.Sequencing results show that L. caseil-lactate dehydrogenase is lacking the N-terminal arm of vertebrate lactate dehydrogenases and electron density maps at 5 Å resolution indicate that ligands might possibly bind in the region of the missing arm. The active site loop is involved in intermolecular contacts and its structure might be different from both, apo- and ternary dogfish l-lactate dehydrogenase.     

Regulation of D-glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe.

D-Glucose-6-phosphate dehydrogenase is a regulatory enzyme of the oxidative pentose phosphate pathway in Schizasaccharomyces pombe. The enzyme is subject to negative cooperative regulation by D-glucose-6-phosphate as characterized by the Hill coefficient of 0.68 +/- 0.04. D-Glyceraldehyde-3-phosphate and D-ribulose-5-phosphate rectify the negative cooperativity as evidenced from a change in the Hill coefficients to 0.98 +/- 0.05 and 1.02 +/- 0.05, respectively. These pentose phosphate pathway intermediates also inhibit the enzyme competitively with respect to D-glucose-6-phosphate. Thus, D-glucose-6-phosphate dehydrogenase provides an avenue for regulating the partitioning of D-glucose between the redundant branches of the oxidative phosphate pathway in S. pombe.     

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.