Isotopic analysis of the reaction catalyzed by glycerol dehydrogenase

Glycerol dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of glycerol to form dihydroxyacetone. Initial velocity, product, and dead-end inhibition studies performed for the forward and reverse reactions support an ordered kinetic mechanism with NAD+ binding first and NADH released last. A monovalent cation is required for enzymatic activity and glycerol binding, with K+ having the highest activity as measured by V. The pH dependence of the kinetic parameters V and V/Kglycerol, as well as the temperature dependence of the V pH profile, suggested that an enzymic carboxylate group functions as a base in catalysis. The pH dependence of the primary deuterium kinetic isotope effect shows that DV/Kglycerol increases from a pH-independent value of 1.15 at high pH values to a pH-independent value of 2.44 at low pH values. DV exhibits a similar pH dependence, increasing from a pH-independent value of 2.57 at high pH values to a pH independent value of 4.88 at low pH values. A chemical mechanism for enzymatic glycerol oxidation is proposed based on the data.     

Diabetes-induced changes in lens antioxidant status, glucose utilization and energy metabolism: effect of DL-alpha-lipoic acid

The study was aimed at evaluating changes in lens antioxidant status, glucose utilization, redox state of free cytosolic NAD(P)-couples and adenine nucleotides in rats with 6-week streptozotocin-induced diabetes, and to assess a possibility of preventing them by DL-alpha-lipoic acid. Rats were divided into control and diabetic groups treated with and without DL-alpha-lipoic acid (100 mg x kg body weight(-1) x day(-1), i.p.). The concentrations of glucose, sorbitol, fructose, myo-inositol, oxidized glutathione, glycolytic intermediates, malate, alpha-glycerophosphate, and adenine nucleotides were assayed in individual lenses spectrofluorometrically by enzymatic methods, reduced glutathione and ascorbate--colorimetrically, and taurine by HPLC. Free cytosolic NAD+:NADH and NADP+:NADPH ratios were calculated from the lactate dehydrogenase and malic enzyme systems. Sorbitol pathway metabolites were found to increase, and antioxidant concentrations were reduced in diabetic rats compared with controls. The profile of glycolytic intermediates (increase in glucose 6-phosphate and fructose 6-phosphate, decrease in fructosel,6-diphosphate, increase in dihydroxyacetone phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and no change in lactate), and 5.9-fold increase in alpha-glycerophosphate suggest diabetes-induced inhibition of glycolysis. Free cytosolic NAD+:NADH ratios, ATP levels, ATP/ADP x inorganic phosphate (Pi), and adenylate charge were reduced in diabetic rats while free cytosolic NADP+:NADPH ratios were elevated. Diabetes-induced changes in the concentrations of antioxidants, key glycolytic intermediates, free cytosolic NAD+:NADH ratios, and energy status were partially prevented by DL-alpha-lipoic acid, while sorbitol pathway metabolites and free cytosolic NADP+:NADPH ratios remained unaffected. In conclusion, diabetes-induced impairment of lens antioxidative defense, glucose intermediary metabolism via glycolysis, energy status and redox changes are partially prevented by DL-alpha-lipoic acid. The findings support the important role of oxidative stress in lens metabolic imbalances in diabetes.     

Malate-aspartate shuttle, cytoplasmic NADH redox potential, and energetics in vascular smooth muscle

The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0. 4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2 consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism.     

Triosephosphate metabolism by mature boar spermatozoa

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.     

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.     

Study of the interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA

Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed. The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806--2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH. A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3'-terminal adenosine and is greater when the 5'-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.     

The presence of rotenone-sensitive NADH dehydrogenase in the long slender bloodstream and the procyclic forms of Trypanosoma brucei brucei

The mitochondrial electron-transport chain present in the procyclic and long slender bloodstream forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in procyclic cells was inhibited 80-90% by antimycin A or cyanide, 15-19% by salicylhydroxamic acid, and 30-35% by rotenone. Cytochrom-c-reductase activity, with proline or glycerol 3-phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome-c-reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone-sensitive NADH dehydrogenase (complex I) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream forms, oxygen uptake with glycerol 3-phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone-sensitive NADH-dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream forms of T. brucei.     

Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously. Nitroxyl is also generated from Angeli's salt (Na2N2O3) at physiological pH, and, indeed, Angeli's salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively. Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angeli's salt. These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred. This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above. The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide. Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s).     

Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos

The sugar-induced inhibition of malolactic fermentation in cell suspensions of Leuconostoc oenos, recently reclassified as Oenococcus oeni (L. M. T. Dicks, F. Dellaglio, and M. D. Collins, Int. J. Syst. Bacteriol. 45:395-397, 1995) was investigated by in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and manometric techniques. At 2 mM, glucose inhibited malolactic fermentation by 50%, and at 5 mM or higher it caused a maximum inhibitory effect of ca. 70%. Galactose, trehalose, maltose, and mannose caused inhibitory effects similar to that observed with glucose, but ribose and 2-deoxyglucose did not affect the rate of malolactic activity. The addition of fructose or citrate completely relieved the glucose-induced inhibition. Glucose was not catabolized by permeabilized cells, and inhibition of malolactic fermentation was not observed under these conditions. 31P NMR analysis of perchloric acid extracts of cells obtained during glucose-malate cometabolism showed high intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate. Glucose-6-phosphate, 6-phosphogluconate, and NAD(P)H inhibited the malolactic activity in permeabilized cells or cell extracts, whereas NADP+ had no inhibitory effect. The purified malolactic enzyme was strongly inhibited by NADH, whereas all the other above-mentioned metabolites exerted no inhibitory effect, showing that NADH was responsible for the inhibition of malolactic activity in vivo. The concentration of NADH required to inhibit the activity of the malolactic enzyme by 50% was ca. 25 microM. The data provide a coherent biochemical basis to understand the glucose-induced inhibition of malolactic fermentation in L. oenos.     

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Fluorescence studies of human semi-beta-hemoglobin assembly

The intrinsic fluorescence properties of human alpha apohemoglobin at protein concentrations from 1 to 5 microM in 0.1 M potassium phosphate buffer, pH 7 or 8 at 5 degrees C were monitored in the absence and presence of a fixed concentration (5 microM) of a fluorescence quenching heme-containing native or Des (146-His, 145-Tyr) beta chain partner. These "reverse quenching" studies revealed that the emission intensity changes observed correlated well with protein concentration and theoretical extent of semi-beta-hemoglobin assembly. Furthermore, the relative quenching efficiencies were calculated to be 0.32, 0.25 and 0.61 for beta (pH 7), beta (pH 8) and Des beta (pH 7) chains, respectively. Thus, heme-mediated quenching was sensitive to the expected pH induced alpha apohemoglobin conformational change and to alteration in beta chain structure. Intramolecular changes induced by carboxylterminal modification (decreased "beta chain self-quenching") appeared to enhance the intermolecular rearrangements (increased "alpha chain partner quenching") seen upon subunit assembly.     

Cytoplasmic redox potential affects energetics and contractile reactivity of vascular smooth muscle

Variations in the cytoplasmic redox potential (Eh) and NADH/NAD ratio as determined by the ratio of reduced to oxidized intracellular metabolite redox couples may affect mitochondrial energetics and alter the excitability and contractile reactivity of vascular smooth muscle. To test these hypotheses, the cytoplasmic redox state was experimentally manipulated by incubating porcine carotid artery strips in various substrates. The redox potentials of the metabolite couples [lactate]/[pyruvate]i and [glycerol 3-phosphate]/[dihydroxyacetone phosphate]i varied linearly (r=0.945), indicating equilibrium between the two cytoplasmic redox systems and with cytoplasmic NADH/NAD. Incubation in physiological salt solution (PSS) containing 10 mm pyruvate ([lact]/[pyr]=0.6) increased O2 consumption approximately 45% and produced anaplerosis of the tricarboxylic acid (TCA cycle), whereas incubation with 10 mm lactate-PSS ([lact]/[pyr]i=47) was without effect. A hyperpolarizing dose of external KCl (10 mM) produced a decrease in resting tone of muscles incubated in either glucose-PSS (-0.8+/-0.8 g) or pyruvate-PSS (-2.1+/-0.8 g), but increased contraction in lactate-PSS (1.5+/-0.7 g) (n=12-18, P<0.05). The rate and magnitude of contraction with 80 mm KCl (depolarizing) was decreased in lactate-PSS (P=0.001). Slopes of KCl concentration-response curves indicated pyruvate>glucose>lactate (P<0.0001); EC50 in lactate (29. 1+/-1.0 mM) was less than that in either glucose (32.1+/-0.9 mm) or pyruvate (32.2+/-1.0 mM), P<0.03. The results are consistent with an effect of the cytoplasmic redox potential to influence the excitability of the smooth muscle and to affect mitochondrial energetics.     

The effect of high sugar intake on the esterification of dihydroxyacetone phosphate by rat liver microsomes

Rat liver microsomes were used as an enzyme source to study dietary-induced changes in the rate of dihydroxyacetone phosphate esterification. Rats were fed (1) 75% glucose or fructose diets for various time intervals, or (2) fed a fractose diet for six days and then a chow diet. Both the glucose and fructose diets produced a 2--3-fold increase in total and neutral glycerolipid formation from dihydroxyacetone phosphate measured in the presence of ATP, palmitate, CoA, and NADH. The increased rate of dihydroxyacetone phosphate esterification and a simultaneous rise in serum triglyceride level in rats fed fructose was rapidly reversed when chow was substituted for the fructose. The results indicate that an increased rate of dihyroxyacetone phosphate esterification may contribute to the acceleration of endogenous glycerolipid biosynthesis noted under these dietary conditions.     

Effect of extracellular AMP on cell proliferation and metabolism of breast cancer cell lines with high and low glycolytic rates

In differentiated tissues, such as muscle and brain, increased adenosine monophosphate (AMP) levels stimulate glycolytic flux rates. In the breast cancer cell line MCF-7, which characteristically has a constantly high glycolytic flux rate, AMP induces a strong inhibition of glycolysis. The human breast cancer cell line MDA-MB-453, on the other hand, is characterized by a more differentiated metabolic phenotype. MDA-MB-453 cells have a lower glycolytic flux rate and higher pyruvate consumption than MCF-7 cells. In addition, they have an active glycerol 3-phosphate shuttle. AMP inhibits cell proliferation as well as NAD and NADH synthesis in both MCF-7 and MDA-MB-453 cells. However, in MDA-MB-453 cells glycolysis is slightly activated by AMP. This disparate response of glycolytic flux rate to AMP treatment is presumably caused by the fact that the reduced NAD and NADH levels in AMP-treated MDA-MB-453 cells reduce lactate dehydrogenase but not cytosolic glycerol-3-phosphate dehydrogenase reaction. Due to the different enzymatic complement in MCF-7 cells, proliferation is inhibited under glucose starvation, whereas MDA-MB-453 cells grow under these conditions. The inhibition of cell proliferation correlates with a reduction in glycolytic carbon flow to synthetic processes and a decrease in phosphotyrosine content of several proteins in both cell lines.     

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.     

The effect of phenazine methosulphate on intermediary pathways of glucose metabolism in the lens at different glycaemic levels

In this study changes in alternative pathways of glucose metabolism are examined in the rat lens using radiolabelled glucose in a 1 hr in vitro incubation of 50 mM or 10 mM glucose with or without 0.1 mM phenazine methosulphate (PMS). PMS which reoxidizes NADPH ensures that the pentose phosphate pathway (PPP) is not limited by the supply of NADP+. The data shows that maximal activation of the PPP (with PMS) is 40% greater at high glucose concentrations than normal glucose. This difference in maximal stimulation may be explained by the increase glucose uptake in the hyperglycaemic incubation. In the high-glucose incubation with PMS, hexokinase activity and the glucose 6-phosphate pool is not limiting for the PPP. Under these conditions, PMS alter the NAD+/NADH and NADP+/NADPH ratio. The change in the redox state alters the flux through the polyol pathway, the glycerol 3-phosphate shuttle and the glycolytic control sites, glyceraldehyde 3-phosphate, pyruvate and lactate dehydrogenases. These results are discussed in relation to hyperglycaemia-induced oxidative stress.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Purification, crystallization and properties of triosephosphate isomerase from human skeletal muscle

1. Triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketoisomerase, EC 5.3.1.1) from human skeletal muscle was purified to homogeneity and crystallized. The crystalline enzyme preparation was resolved on polyacrylamide-gel electrophoresis into three isoenzymes. 2. The molecular weight of the enzyme estimated by gel filtration method was found to be 57,400 +/- 3000. Molecular weight determination under dissociation conditions indicated a dimeric subunit structure of the enzyme. 3. The apparent Km for D-glyceraldehyde-3-phosphate as substrate is 0.34 mM, and for dihydroxyacetone phosphate, 0.61 mM. Vmax of the reaction is, respectively, 7200 and 660 units/mg protein at 25 degrees C and pH 7.5. 4. Molecular and kinetic properties of triosephosphate isomerase from human skeletal muscle are very similar to those of rabbit muscle enzyme.