The oxaloacetate reductase activity of vertebrate lactate dehydrogenase

• 1.1. Lactate dehydrogenase is able to catalyse the reduction of oxaloacetate utilizing NADH as coenzyme but, contrary to a previous report, with greatly reduced values for both K_m and V_max when compared to the normal substrate pyruvate.
• 2.2. A modification to the published procedure for the purification of vertebrate l-lactate dehydrogenase by affinity chromatography on oxamated Sepharose is described.
• 3.3. Supernatant malate dehydrogenase does not catalyse the reduction of pyruvate at rates greater than 1%, of the rates with oxaloacetate.

     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.

     

Kinetic mechanism of guinea-pig skeletal muscle lactate dehydrogenase (M4) with oxaloacetate-NADH and pyruvate-NADH as substrates

• 1.1. Guinea-pig skeletal muscle lactate dehydrogenase M₄ isoenzyme, with pyruvate and NADH as substrates, is adapted to an ordered bi-bi ternary complex mechanism at pH 7.0.
• 2.2. In the same conditions, the kinetic mechanism of the reaction, with oxaloacetate and NADH as substrates, is of the rapid equilibrium ordered bi-bi ternary complex type; NADH is the first substrate in the reaction sequence.

     

Analysis of the effect of light on carbohydrate metabolism in Phycomyces sporangiophores and mycelia

• 1.1. The pattern of the changes shown for the metabolic intermediates studied from two structures is not coincident.
• 2.2. Both in sporangiophores and mycelia we found very low pyruvate/lactate and oxaloacetate/malate ratios.
• 3.3. The effect of light on the whole Phycomyces was evaluated. From this, we cannot exclude a dependence on the gene mad B and/or car S products from the effect found in the level of glucose-6-phosphate, l-alanine, l-malate and oxaloacetate.

     

Lactate dehydrogenase isozymes of the leopard danio,Brachydanio nigrofasciatus: their characterization and ontogeny

• 1.1. The tissue specific patterns and ontogeny of lactate dehydrogenase (LDH) are reported.
• 2.2. While all tissues (eye, brain, heart, intestine, liver, ovary and skeletal muscle) show isozymes of A and B subunit composition, only liver extracts possess isozymes resulting from C subunit synthesis.
• 3.3. The A₄ homopolymer appears simultaneously with initial muscle contractility and is correlated with the physiological function of muscular contraction.
• 4.4. The activation of the Ldh-C locus is correlated with the first functioning of liver. It is suggested that the state of differentiation of liver cells may be the stimulus required for C locus expression.

     

Effect of adenosine on gluconeogenesis in the perfused liver of chicken and guinea-pig

• 1.1. Glucose formation from lactate by the perfused liver of 48 hr starved chickens was strongly inhibited by adenosine (Ado); the half-maximal inhibition was attained at 40 μM. This effect was paralleled by a four- to five-fold increase of ATP content as determined in freeze-clamped liver.
• 2.2. In chicken liver homogenate gluconeogenesis from precursors such as alanine, glutamate, glutamine and aspartate, which are not converted into glucose by the perfused chicken liver, proceeded at rates equal to or higher than that with lactate, being markedly inhibited by Ado.
• 3.3. In the perfused guinea-pig liver glucose synthesis with lactate, propionate, glycerol and fructose was also inhibited by Ado; however, when precursors such as pyruvate, glutamine and a mixture of lactate + pyruvate were supplied to the liver Ado did not inhibit gluconeogenesis.
• 4.4. Assay of adenine nucleotides in the perfused guinea-pig liver, stopped by freeze-clamping technique in a number of experimental variants, revealed no correlation between the rate of gluconeogenesis and the changes induced by Ado in the adenine nucleotide pool.
• 5.5. In the perfused liver of both chicken and guinea-pig Ado produced an increase of the lactate to pyruvate ratio and, in general, a diminution of the content of malate-aspartate shuttle intermediates.
• 6.6. The results are interpreted as suggesting that the inhibitory effect of Ado on hepatic gluconeogenesis is not necessarily mediated by the changes in the adenine nucleotide pool.

     

Novel amino acid linked dehydrogenase in the sponge Halichondria panicea (pallas)

• 1.1. The sponge Halichondria panicea has a complete sequence of glycolytic and tricarboxylic acid cycle enzymes.
• 2.2. However, there is no detectable lactate dehydrogenase in H. panicea and lactate dehydrogenase appears to be functionally replaced by an enzyme which catalyses the reductive condensation of pyruvate and glycine to yield 2-methylimino-diacetic acid (strombine).
• 3.3. The intracellular distribution and kinetic properties of this novel enzyme (strombine dehydrogenase) have been investigated and its role in metabolism is discussed.

     

Light-induced effects of several enzymes of carbohydrate metabolism in Phycomyces blakesleeanus

• 1.1. The photoregulation shown by glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase appears to be independent of the mad gene product(s) and also independent of carotene biosynthesis regulation.
• 2.2. The photoregulation of malate dehydrogenase appeared to be dependent on the mutation of the mad and car S genes.
• 3.3. Pyruvate kinase and lactate dehydrogenase may be classified as light-independent.
• 4.4. The action of ATP and fructose 1,6-bisphosphate on the enzymes studied was generally independent of light/dark grown conditions.
• 5.5. However, the effect of fructose 1,6-bisphosphate on Phycomyces pyruvate kinase appears to be light-dependent.

     

Distribution of opine dehydrogenases and lactate dehydrogenase activities in marine animals

• 1.1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence.
• 2.2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata.
• 3.3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated.
• 4.4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.

     

Shift in vivo in the lactate dehydrogenase isoenzyme pattern in experimental models conditioning the metabolic adaptation of rat brain

• 1.1. In order to analyse the possible shift in the lactate dehydrogenase isoenzyme pattern, experimental models affecting the glycolytic pathway are developed, including phenylalanine, thiamine and epinephrine administration, and a vitamin free diet.
• 2.2. The results obtained show that a shift in the lactate dehydrogenase isoenzyme pattern occurs in every change in physiological condition.
• 3.3. These shifts are in accordance with the kinetic properties of each isoenzyme and account for a metabolic adaptation to each physiological state.

     

Kinetic mechanism of the molecular forms of chicken liver mitochondrial malate dehydrogenase

• 1.1. The reaction kinetic mechanism (pH 7.4) of the molecular forms of chicken liver m-MDH is of the ordered bi-bi ternary complex type with the existence of the E-oxaloacetate, E-L-malate, E-NAD⁺ oxaloacetate, E-NADH-l-malate, E-NAD⁺-NADH, E-NAD⁺-NAD⁺, E-NADH-NAD⁺ and E-NADH-NADH abortive complexes.
• 2.2. The saturating concentration values of the substrates are notably modified, in certain cases, in the presence of the reaction products.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Thermal stability of the molecular forms of guinea-pig skeletal muscle cytoplasmic malate dehydrogenase and kinetic mechanism of the thermostable form

• 1.1. Guinea-pig skeletal muscle cytoplasmic malate dehydrogenase appears under two molecular forms; the heating of the dialyzed soluble fraction of the tissue shows that the A form is stable at 55 C, while the B form is inactivated. Under these conditions, the lactate dehydrogenase M₄ isoenzyme becomes considerably unstable; nevertheless, its activity is notably preserved with NADH.
• 2.2. The reaction kinetic mechanism of the isolated A form has been determined (pH 7.4), enabling the nature of the abortive complexes and the values of K_eeq and of ΔG°′ of the reaction to be determined.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Alcohol dehydrogenase ontogeny and liver maturation in the leopard danio,brachydanio nigrofasciatus

• 1.1. The tissue specificity and ontogeny of alcohol dehydrogenase (ADH) are reported for the leopard danio, Brachydanio nigrofasciatus.
• 2.2. Of the seven adult tissues assayed (eye, brain, kidney, liver, ovary, skeletal muscle and stomach), only liver extracts showed ADH activity.
• 3.3. The activation of the Adh locus is correlated with the first functioning of liver. It is suggested that the state of liver cell differentiation may be the stimulus necessary for Adh locus expression.

     

A one-step pmr determination of hydrogen transfer stereospecificity of NADP+-linked oxidoreductases

• 1.1. A simple, facile one-step method has been devised to measure the stereospecificity of NADP⁺-linked oxidoreductases. The procedure involves coupling the test enzymes to enzymes of known stereospecificity in the presence of deuterated substrates. The regenerated NADP⁺ in the coupled reactions is analyzed by PMR for its deuterium content at the carbon-4 position of the nicotinamide ring.
• 2.2. It is found that malate dehydrogenase (EC 1.1.1.37). lactate dehydrogenase (EC 1.1.1.27) and glycerate dehydrogenase (EC 1.1.1.29) are A-side stereospecific whereas glutamate dehydrogenase (EC 1.4.1.3) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) are B-side stereospecific.
• 3.3. Enzymes which can utilize both NAD⁺ and NADP⁺ have the same stereospecificity with respect to the coenzyme.

     

Malate dehydrogenase and non-specific esterase isoenzymes of eggs of the honey bee (Apis mellifera L.)

• 1.1. Isoelectric focusing on polyacrylamide gels was used to detect malate dehydrogenase and non-specific esterase isoenzymes in diploid honey bee (Apis mellifera L.) eggs of several known ages.
• 2.2. It was determined that the number of MDH and EST isoenzymes increased as development proceeded with an apparent increase in the quantity of some of the isoenzymes.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Glycolytic enzymes of fowl and turkey spermatozoa

• 1.1. It was confirmed that, under anaerobic conditions, fowl spermatozoa formed lactate from glucose thirteen times faster than turkey spermatozoa.
• 2.2. The profiles of glycolytic enzyme activities were similar for spermatozoa from both species; however fowl spermatozoal activities were generally 2- to 4-fold higher.
• 3.3. Exceptions were glycerophosphate mutase and lactate dehydrogenase activities which were respectively 9.5 and 41 times greater in fowl spermatozoa.
• 4.4. In both species, spermatozoal glyceraldehyde-3-phosphate dehydrogenase had the lowest activity of the glycolytic enzymes.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.