Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.     

Pyruvate kinase in the bovine adrenal cortex

Pyruvate kinase from bovine adrenal cortex was purified to an electrophoretically homogeneous state. The molecular weight of the native enzyme is about 230 000, that of one subunit is 57 000. The maximal values of the pyruvate kinase initial reaction rate were obtained in 50 mM imidazole-acetate buffer within the pH range of 6.8 to 7.0. The curve of the initial pyruvate kinase reaction rate versus phosphoenolpyruvate (PEP) and ADP concentrations is hyperbolic and obeys the Michaelis-Menten kinetics with Km for PEP and ADP of 0.055 X 10(-3) M and 0.25 X 10(-3) M, respectively. The enzyme is activated by Mn2+ and Co2+ by 43 and 38%, respectively. IDP, GDP, and UDP may be used as analogs of ADP. The enzyme is not activated by fructose-1.6-diphosphate and is inhibited by L-phenylalanine and ATP.     

The effects of pH and temperature on the kinetic properties of skeletal muscle lactate dehydrogenase from anuran amphibians

Summary Muscle LDH activities were measured in two anuran amphibians with different behaviour and ecology, Rana perezi and Bufo calamita. Both pyruvate reduction and lactate oxidation were measured at temperatures of 15, 20 and 30°C, and at pH 7.0, 7.4, and 8.0. Pyruvate and lactate muscle concentrations were determined in individuals at rest and after exercise. R. perezi muscle used anaerobic glycolysis during 3 min of exhaustive exercise, with rising pyruvate and lactate concentrations. Enforced walking for 30 min caused high variability in lactate concentration in B. calamita muscle. Temperature and pH changes affected apparent Km values for pyruvate. When these factors varied simultaneously, enzyme affinity tended not to change. Thus, the thermodynamic effect on pyruvate reduction activity is high, especially at physiological substrate concentrations. In contrast, lactate oxidation activity tended to stabilize when temperature and pH varied jointly. Inhibition by substrate, pyruvate or lactate, seemed to have no importance in vivo.During exercise there was a rise in pyruvate concentration, and a probable decrease in pH, which increased pyruvate reduction reaction and decreased lactate oxidation, contributing to lactate accumulation in Rana perezi muscle. B. calamita muscle did not show pyruvate increase after exercise and its LDH was less dependent on pH at physiological concentrations. Pyruvate reduction rate did not therefore increase. R. perezi muscle enzyme had features of anaerobic LDH while B. calamita LDH muscle was more similar to mammalian heart enzyme, with differences in accordance with the different behaviour of these anurans.Abbreviations LDH lactate dehydrogenase     

Regulation of lactate dehydrogenase and change of fermentation products in streptococci.

Streptococcus mutans JC 2 produced mainly lactate as a fermentation product when grown in nitrogen-limited continuous culture in the presence of an excess of glucose and produced formate, acetate, and ethanol, but no lactate, under glucose-limited conditions. The levels of lactate dehydrogenase (LDH) in these cultures were of the same order of magnitude, and the activity of LDH was completely dependent on fructose-1,6-diphosphate (FDP). The intracellular level of FDP was high and the level of phosphoenolpyruvate (PEP) was low under the glucose-excess conditions. In the glucose-limited cultures, all glycolytic intermediates studied, except PEP, were low. S. mutans FIL, which had an FDP-independent LDH and similar levels of glycolytic intermediates as S. mutans JC2, produced mainly lactate under glucose-excess or under glucose-limited conditions. LDH of Streptococcus bovis ATCC 9809 was dependent on FDP for activity at a low concentration of pyruvate but had a significant activity without FDP at a high concentration of pyruvate. This strain also produced mainly lactate both under glucose-excess and glucose-limited conditions. The levels and characteristics of these LDHs were not changed by the culture conditions. These results indicate that changes in the intracellular level of FDP regulate LDH activity, which in turn influences the type of fermentation products produced by streptococci. PEP, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and inorganic phosphate significantly inhibited LDH activity from S. mutans JC 2 and may also participate in the regulation of LDH activity in other streptococci.     

Enzymic activities of carbohydrate,purine, and pyrimidine metabolism in the Anaeroplasmataceae (class Mollicutes)

Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161^T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PP_i-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PP_i-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P ₂). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P ₂), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PP_i-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.Abbreviations EMP Embden-Meyerhof-Parnas - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PEP phosphoenolpyruvate - PFK phosphofructokinase - PPDK pyruvate, orthophosphate dikinase - TCA cycle tricarboxylic acid cycle Note: Other abbreviations used are as per the instruction to authors, or the reference cited therein (Eur J Biochem 1:259), or Biochem J 120:449 (which supercedes a portion of the first reference)     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis

1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of microM concentrations of fructose-1,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.     

Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium.

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.     

Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Characterization of the kinase activity of bovine adrenal pyruvate dehydrogenase complex

In the presence of [gamma-32P]ATP the bovine adrenal pyruvate dehydrogenase complex accepts the label simultaneously and becomes inactivated. This suggests the existence of kinase in the composition of the complex as is typical of the complexes from other animal sources. The Pi is incorporated into the subunit with molecular weight of 42 000. The kinase activity of the adrenal pyruvate dehydrogenase complex is high: within the first 20 sec of incubation with ATP the inactivation is as high as 60%. The pH optimum for kinase is around 7.3. The apparent Km value for ATP with 50 mM KCl is 7 microM; that in the absence of KCl is 10 microM. ADP is a competitive inhibitor of kinase with respect to ATP (Ki = 100 microM), when K+ are present in the medium. Thiamine pyrophosphate and pyruvate decrease the rate of pyruvate dehydrogenase complex inactivation.     

Lactate dehydrogenases in Antarctic and temperate fish species

1. The kinetics of lactate dehydrogenase (both forward and back reaction) in cardiac and skeletal muscle of an Antarctic teleost have been compared with a temperate teleost of comparable morphology and ecology. 2. In both species the forward reaction (pyruvate to lactate) is maximally activated at 2.5-4 mM pyruvate and inhibited above this level. 3. The Michaelis constant (Km) for pyruvate is not significantly different between muscle types or between species when measured at their normal environmental temperature. 4. Km for pyruvate varies with temperature in a positive direction. 5. The back reaction (lactate to pyruvate) is maximally activated by 12-16 mM lactate but only in skeletal muscle of the antarctic species is there inhibition above this level. 6. The Km for lactate is significantly (P less than 0.05) lower in the Antarctic fish cardiac muscle. 7. While the two species are morphologically and ecologically similar, differences at the biochemical level are discussed with respect to environmental temperature range and conservation of enzymic characteristics.     

Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion

Under anaerobic conditions, competition for pyruvate between the branch point enzymes pyruvate formate lyase (PFL, Km = 2 mM) and fermentative lactate dehydrogenase (LDH, Km = 7.2 mM) determines the partition of carbon flux. Two Escherichia coli mutant strains, one deficient in ackA, pta, and ldhA and the other overexpressing LDH, were constructed to systematically analyze the effects of these perturbations in the existing pathways on the redistribution of carbon fluxes. Deletion of the lactate and acetate synthesis pathways was detrimental to cell growth. Carbon flux is forced through ethanol and formate production pathways, resulting in a concomitant increase in those fluxes. In addition, overexpression of LDH simultaneously increases the common flux as well as the flux to the competing acetyl-CoA branch. Overexpression of lactate dehydrogenase (ldhA) in the parent strain increases the lactate synthesis rate from 0.19 to 0.40 mmol/g-biomass-h when the LDH activities increases from 1.3 to 15.3 units. Even an increase of more than 10 times in the LDH activity fails to divert a large fraction of the carbon flux to lactate; the majority of the flux still channels through the acetyl-CoA branch. Overexpression of LDH in the parent strain simultaneously increases the common flux as well as the flux through the acetyl-CoA branch. Subsequently, the flux amplification factors (or deviation indices which can be related to the flux control coefficients) are positive for all three fluxes occurring at the pyruvate node.     

Lactate dehydrogenase isoenzymes in the eye, cardiac and skeletal muscles of several decapods]

Properties of lactate dehydrogenase (LDH) in the eye, heart and muscles of Hemigrapsus sanguineus, Paralithodes camtschatica, Erimacrus isenbeckii, Pandalus latirastrus, Pagurus brachiomastus have been studied with acrylamide gel electrophoresis and kinetics analysis. LDH in all the tissues of all the representatives studied was found to be specific for L-pyruvate and lactate; it migrated in electrophoresis as a single band revealing low mobility towards anode. The isoenzyme from P. camtschatica and P. latirastrus differed from the isoenzymes of other animals studied by higher mobility towards anode that reflected higher negative value of its total charge. The LDH isoenzymes in all the animals studied resembled the A4 (LDH5) of the vertebrates being unstable to the denaturing action of high temperature and being unaffected by high concentrations of pyruvate up to 1.0.10-3M. On the other hand, in conrast to the A4 of mammals, the LDH in question displayed enhancement of the reaction rate and decrease of the Km values upon increase in the NAD+ and NAD.H concentrations both in the presence of high or low lactate and pyruvate concentrations. The isoenzymes displayed catalytic activity also in the presence of NADP, the Km values for pyruvate in the presence of equimolar (2.25 mM) concentrations of NAD.H or NADP.H were practically identical and were found to be within the limits of 14-26.10-5 M. Molecular weight of the LDH studied assessed by the gel filtration method was found to be 130-140,000. It is suggested that the LDH isoenzyme from the representatives of the decapod crayfish studied is homologous in its certain properties to the homotetrameric A4 form of the vertebrates.     

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

Pyruvate dehydrogenase and the path of lactate degradation in Desulfovibrio vulgaris Miyazaki F

Pyruvate dehydrogenase from Desulfovibrio vulgaris Miyazaki F was partially purified from the soluble fraction of the bacterial sonicate, and characterized. The enzyme catalyzes oxidative decarboxylation of pyruvate to produce acetyl-CoA, in contrast to statements in current review articles in which acetyl phosphate is indicated to be a direct decomposition product of pyruvate in sulfate-reducing bacteria. The established reaction stoichiometry is: pyruvate + CoA + FMN----acetyl-CoA + CO2 + FMNH2. The Km values are 2.9 mM for pyruvate, 32 microM for CoA and 6.7 mumol for FMN. Participation of thiamine diphosphate in the enzymic process was not proven. 2-Oxobutyrate, but not 2-oxoglutarate, can substitute for pyruvate. The three flavin compounds, FMN, FAD, and flavodoxin, as well as clostridial ferredoxin, serve as electron carriers for the enzyme. Thus the enzyme is a kind of pyruvate synthase [EC 1.2.7.1], but acts in the direction of pyruvate degradation in the growing cells. The rate of cytochrome C3 reduction is extremely low, but in the presence of flavodoxin as an electron mediator, the reduction rate of cytochrome C3 becomes faster than the reduction rate of flavodoxin alone. It seems that the physiological electron acceptor for this enzyme is flavodoxin, which might be complexed with cytochrome C3 to produce a very efficient electron transfer system in the cell. The soluble fraction of D. vulgaris cells has been proved to contain, in addition to the pyruvate dehydrogenase, lactate dehydrogenase (Ogata, M., Arihara, K., & Yagi, T. (1981) J. Biochem. 89, 1423-1431), phosphate acetyltransferase and acetate kinase, i.e., all the enzymes necessary to convert lactate to acetate, producing ATP by substrate level phosphorylation.     

Creatine kinase equilibrium and lactate content compared with muscle pH in tissue samples obtained after isometric exercise

Muscle biopsies taken from the musculus quadriceps femoris of man were analysed for pH, ATP, ADP, AMP, creatine phosphate, creatine, lactate and pyruvate. Biopsies were taken at rest, after circulatory occlusion and after isometric contraction. Muscle pH decreased from 7.09 at rest to 6.56 after isometric exercise to fatigue. Decrease in muscle pH was linearly related to accumulation of lactate plus pyruvate. An increase of 22mumol of lactate plus pyruvate per g of muscle resulted in a fall of 0.5pH unit. The apparent equilibrium constant of the creatine kinase reaction (apparent K(CK)) increased after isometric contraction and a linear relationship between log(apparent K(CK)) and muscle pH was obtained. The low content of creatine phosphate in muscle after contraction as analysed from needle-biopsy samples is believed to be a consequence of an altered equilibrium state of the creatine kinase reaction. This in turn is attributed mainly to a change in intracellular pH.     

beta-Nicotinamide-alpha-adenine dinucleotide. Synthesis and properties of a coenzyme analog.

It was not possible to synthesize beta-nicotinamide-alpha-adenine dinucleotide (NalphaAD) via the NAD pyrophosphorylase-catalyzed reaction of beta-nicotinamide mononucleotide and alpha-adenosine-5'-O-triphosphate. It was therefore prepared by reacting a mixture of beta-nicotinamide mononucleotide and alpha-adenosine-5'-O-monophosphate in aqueous pyridine, using N,N'-dicyclohexylcarbodiimide as condensing agent. NalphaAD can replace NAD in the lactate-dehydrogenase-catalyzed oxidation of lactate to pyruvate with LDH 4M from hog muscle as well as LDH 4H from pig heart. Km values are found to be about one order of magnitude higher for NalphaAD than for NAD. The turnover number for NalphaAD with LDH 4H is similar to that of NAD (95%), whereas it is reduced to one third (35%) in the reaction with LDH 4M. Experiments findings are discussed on the lines of a NAD-equivalent conformation of NalphaAD in the holoenzyme complex.