H4-isozyme of lactate dehydrogenase in the solution of sodium chloride--3. The enzymatic activity and the pyruvate inhibition

1. Low enzymatic activities in low pyruvate concentrations and high Km were observed in sodium chloride solutions. 2. The pyruvate inhibition shown by the % activity at 1 mM pyruvate was lower sodium chloride than in 0.1 M sodium phosphate. 3. At 40 degrees C, as compared with results at 20 degrees C, less pyruvate inhibition was observed in phosphate buffer and in sodium chloride solutions. 4. By using the equilibrium constants between dimer and tetramer, a theoretical explanation is proposed for the pyruvate inhibition. In this explanation, it is suggested that the quaternary complex which is composed of tetrameric enzyme, coenzyme and two kinds of pyruvates was the main cause of the pyruvate inhibition.     

Characterization of NADP+: 3 beta-hydroxysteroid dehydrogenase from microsomes of rat liver

A NADP(+)-dependent 3 beta-hydroxysteroid dehydrogenase activity was localized in the microsomal fraction of rat liver. This enzyme was solubilized and separated completely from 3 alpha-hydroxysteroid dehydrogenase by Matrex red A column chromatography. Partially purified 3 beta-hydroxysteroid dehydrogenase catalyzed the oxidation and reduction between the 3 beta-hydroxyl and 3-ketonic group of steroids or bile acids having no double bond in the A/B ring, but was inactive toward 3 alpha-hydroxyl group. The enzyme required NADP+ for oxidation and NADPH for reduction. The activity was inhibited by p-chloromercuribenzoic acid or p-chloromercuribenzenesulfonic acid at the concentration of 10(-4) M. The molecular weight of the enzyme was estimated to be about 43,000 by Sephadex G-200 column chromatography. From these results, it is concluded that the enzyme is a new type of microsomal NADP+:3 beta-hydroxysteroid dehydrogenase.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Purification and properties of l-lactate dehydrogenase from Acinetobacter calcoaceticus

Abstract Membrane-bound l -lactate dehydrogenase has been purified almost to homogeneity from Acinetobacter calcoaceticus . The enzyme is an oligomeric protein of sub-unit M _r 40 000 containing non-covalently bound FMN as a prosthetic group. Purified l -lactate dehydrogenase has an apparent K _m of 83 μM for l -lactate but has no activity with, and is not inhibited by, d -lactate. The enzyme is strongly inhibited by HgCl₂, but other thiol reagents and metal-chelating compounds have little or no effect upon its activity.     

Regulation of lactate dehydrogenase activity in Rothia dentocariosa by fructose 1,6-diphosphate and adenosine 5''-triphosphate.

The L-(+)-lactate dehydrogenase from Rothia dentocariosa strain 17931 is activated by fructose 1,6-diphosphate and inhibited by adenosine 5'-triphosphate. The enzyme has a molecular weight of 120,000. In these respects, it resembles the lactate dehydrogenase of Actinomyces viscosus.     

Purification and characterization of N-methylalanine dehydrogenase.

Cell free extracts of Pseudomonas MS previously have been shown to carry out the synthesis of a novel amino acid, N-methylalanine (Kung, H.F., and Wagner, C. (1970) Biochim. Biophys. Acta 201, 513-516). An enzyme has been isolated from this organism which is responsible for the synthesis of N-methylalanine. The stoichiometry of the reaction catalyzed by this enzyme leads to the following formulation: Methylamine + pyruvate + NADPH + H-+ yields N-methylalanine + NADP-+ + H2O. This enzyme has been physically separated from alanine dehydrogenase, which is also present in these extracts. This new enzyme has been named N-methylalanine dehydrogenase. It has been purified to near homogeneity as judged by disc gel electrophoresis. Gel filtration chromatography showed that N-methylalanine dehydrogenase has an apparent molecular weight of 77,000, while electrophoresis in sodium dodecyl sulfate gave rise to a single band with a molecular weight of approximately 36,500. The enzyme is optimally active in the pH range between 8.2 and 8.6. The apparent K-m values for pyruvate, NADPH, and methylamine, respectively, are 1-5 times 10 minus 2 M, 3-5 times 10 minus 5 M, and 7.5 times 10 minus 2 M.     

Partial purification from human mononuclear cells and placental plasma membranes of an insulin mediator which stimulates pyruvate dehydrogenase and suppresses glucose-6-phosphatase

A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G-6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction. This material was partially purified by molecular-exclusion chromatography, ion-exchange chromatography, and hydroxylapatite chromatography. This was found to stimulate pyruvate dehydrogenase and inhibit glucose-6-phosphatase in a dose-dependent manner. The amount or ability of this substance to stimulate pyruvate dehydrogenase was increased in the proportion to the concentration of insulin. The stimulation of pyruvate dehydrogenase by the factor was eliminated when sodium fluoride was presented in the assay of the activation. This result implied that the activation of pyruvate dehydrogenase was mediated by the stimulation of the phosphatase of pyruvate dehydrogenase complex. Each material isolated from insulin-treated human placental plasma membranes and mononuclear cells shared a number of important characteristics of putative second messengers of insulin action as follows: (i) heat and acid stability; (ii) a similar molecular weight; (iii) increased activity of pyruvate dehydrogenase in a insulin-dependent manner; and (iv) stimulated pyruvate dehydrogenase by the sodium fluoride-sensitive mechanism. This human putative second messenger of insulin action was eluted from the anion-exchange resin AG1-X8 at an ionic strength of 3–4 m, as well as from the hydroxylapatite column at a phosphate concentration of 2–3 m.     

Thermophilic NAD-dependent glutamate dehydrogenase from Bacillus stearothermophilus

A rapid purification procedure for glutamate dehydrogenase (GDH) from Bacillus stearothermophilus var calidolactis was developed. The homogeneous enzyme with a total molecular weight of approximately 240,000 daltons, contained 6 identical subunits. No high molecular weight form of GDH present in crude extracts was found after elution of the enzyme from a 5'AMP-Sepharose column with 4 M urea. The purified enzyme functions in both directions i.e. amination and deamination and is strictly specific for NAD. 2-Oxo glutarate, glutamate or 2-mercaptoethanol protects against heat inactivation. NADH or ammonia, on the other hand, makes GDH more sensitive to heat. The purified enzyme undergoes thermal inactivation process.     

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.     

Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.     

H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea.

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.     

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.     

Intracellular lactate dehydrogenase concentration as an index of cytotoxicity in rat hepatocyte primary culture

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC₅₀ values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.Abbreviations DMSO dimethyl sulfoxide - HPC hepatocyte primary culture - IC₅₀ 50% inhibitory concentration - LDH lactate dehydrogenase     

Purification and properties of human hepatic 3 alpha-hydroxysteroid dehydrogenase.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5 beta-[3H]dihydrocortisol (5 beta-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6-5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3 alpha-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5 alpha- and 5 beta-DHF and 5 alpha-dihydrotestosterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8-7.4. The apparent Km for 5 beta-DHF was 18 microM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4 degrees C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3 alpha-HSD from human tissue.     

Purification and properties of a fructose-1,6-diphosphate activated L-lactate dehydrogenase from Staphylococcus epidermidis.

L-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 Yielded molecular weights of about 130 000 and 126 000, respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68 000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.     

Regularities in the kinetic of photoactivation of lactate dehydrogenase by the action of UV light in different microenvironment

The kinetics of photoinactivation of cardiac (H4) and muscular (M4) isoforms of lactate dehydrogenase irradiated by UV light (240-390 nm) in the free form and in the presence of sodium azide, D-mannitol, and serotonin was studied. It was shown that the decrease in the catalytic activity of both enzymes can be described by the kinetics of the first-order monomolecular reaction. The inactivation rate constant of lactate dehydrogenase M4 is considerably higher than that of lactate dehydrogenase H4, indicating a greater photochemical lability of the isoform M4. It was shown that sodium azide has a different protective action on the proteins studied. The irradiation of the muscular isoform in the presence of serotonin and D-mannitol did not change the character of the "dose-effect" curve and only led to a decrease in the photoinactivation rate constant of the protein.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE-cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min^-1 (molecule of GDH)^-1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP-GDH is composed of six identical polypeptidic chains.
The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP-GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2-mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 m M for α-ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its K_m value increased from 2.6 to 21.2 m M when the ammonium concentration exceeded 16 m M . The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the K_m values, indicate that the NADP-GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.     

Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils

Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD+ and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD+ and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for omega-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes omega-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1:2. The affinity of the dehydrogenase for 20-OH-LTB4 may be higher than that for 12-hydroxylaurate (Km = 70 microM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase.     

Effect of vacuolization on the sodium concentration in an isolated muscle fiber]

Using the Perkin Elmer flame photometer sodium and potassium concentrations have been measured in muscle fibers from the m. ileofibularis of Rana temporaria. After 30 minutes preincubation in the Ringer solution, made hypertonic by the addition of 0.22M glycerol, the muscle fibers were incubated in the normal Ringer solution for 30 min. These fibers showed a vacuolation and an increase in total fiber sodium up to 37.2 mmol/l +/- 5.9 S. E., or 45.8 mmol/kg H2O +/- 7.3 S. E. No significant changes in potassium concentration were observed. Then, the fibers were exposed again to the Ringer solution containing 0.22 M glycerol. This procedure caused the disappearance of vacuoles and decrease in fiber sodium concentration down to 17.7 mmol/l +/- 1.6 S. E., or 21.8 mmol/kg H2O +/- 2.0 S. E. The effect of vacuolation was not blocked by ouabain (1.10(-4) M). It is suggested that the vacuoles have a high NaCl concentration. A model for NaCl and water accumulation in T-tubules is presented.     

Adsorptive pinocytosis of 125I-labelled lactate dehydrogenase isoenzymes H4 and M4 by rat yolk sacs incubated in vitro.

The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.