NAD Oxidoreductase Research Articles

Coupled optical rate determinations of amino acid oxidase activity

Methods are described in which liberation of ammonia from amino acid substrates by the d- and l-amino acid oxidases may be coupled with the NADH-dependent reductive amination of 2-oxoglutarate catalysed by exogenous glutamate dehydrogenase ( l-glutamate: NAD oxidoreductase (deaminating), EC 1.4.1.2). The inhibitin of d-amino acid oxidase ( d-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) by ADP needed to activate and stabilise glutamate dehydrogenase was relieved by FAD, and the substrate was d-alanine at approximately 6-fold K m concentration. Neither FAD or FMN were required in the l-amino acid oxidase ( l-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.2) assay; this utilised l-leucine as substrate in a concentration approximately 7-fold the K m value. The methods were reasonably sensitive and precise, and a linear relationship between activity and enzyme concentration prevailed up to an absorbance change of 0.050 per min. They have the advantage of being amenable to automation and to employment of fluorescence techniques should greater sensitivity be required.

3alpha-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni: Kinetic Properties with NAD and Its Thionicotinamide Analogue

The kinetics of 3alpha-hydroxysteroid : NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3alpha-hydroxysteroids from both the C19, C21, and C24 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11beta-hydroxy or an 11-oxo group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3alpha-hydroxy group. No such effect could be seen when the 11-hydroxy group was in the alpha-position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration-depenedent monomeric-dimeric transition of the 3alpha-hydroxysteroid dehydrogenase.

Steroidogenesis in rabbit preimplantation embryos.

Rabbit preimplantation embryos were flushed from the reproductive tract at 24 hr (1- to 2-cell stage), 48 hr (morula), 72 hr (morula), 96 hr (blastocyst), 120 hr (blastocyst), and 144 hr (blastocyst) post coitum. At 168 hr (early postimplantation period), gestation sacs were excised, frozen, and sectioned in a cryostat. Delta5-3beta-Hydroxysteroid dehydrogenase [3(or 17)beta-hydroxysteroid:NAD(P) oxidoreductase, EC 1.1.1.51] activity was determined histochemically in whole preimplantation embryos and in sectioned postimplantation embryos. 3beta-Hydroxysteroid dehydrogenase activity began at 48 hr and was sustained through the late blastocyst stage (144 hr), with the exception of a brief drop, possibly cessation, of activity at 72 hr. There was no activity at 168 hr. Since 3 beta-hydroxysteroid dehydrogenase is a key enzyme in the metabolism of steroid hormones, its presence is strong evidence for steroidogenesis. Only 144-hr preimplantation embryos were used to determine 17 beta-hydroxysteroid dehydrogenase (estradiol-17beta:NAD 17-oxidoreductase, EC 1.1.1.62) activity, which was present, suggesting synthesis of estrogen. By means of radioimmunoassay, 144-hr preimplantation embryos were found to contain estradiol-17beta. Other authors have shown that rabbit blastocysts contain progesterone and other steroids, and these embryos can synthesize steroids from non-steroid and steroid precursors. Therefore, our results plus those of others prove that rabbit preimplantation embryos synthesize steroid hormones. Our present and previous results (with rats, hamsters, and mice) suggest that the steroid hormones synthesized by the embryo are critical for preimplantation embryogenesis and for implantation of the lbastocyst.

Purification and properties of malic dehydrogenase from Trichomonas gallinae

The malic dehydrogenase (MDH 2, l-malate: NAD oxidoreductase, E.C. 1,1.1.37) of Trichomonas gallinae was purified 215-fold and characterized. The molecular weight was found to be 72,000 and the enzyme protein contained essential cations and sulfhydryl groups. Polyacrylamide gel electrophoresis before and after extensive purification yielded a single band of malic dehydrogenase activity strongly suggesting only one molecular form of the enzyme. Analysis of kinetic data yielded the following K m values: oxalocetate, 16 μM; malate, 200 μM; NADH 11 μM; and NAD, 70 μM. The enzyme was absolutely specific for l-malic acid, NAD, and NADH. The enzyme exhibited a broad band of heat stability with an optimum of 51 C. The pH optimum in the direction of oxalacetate reduction was 9.0. The pH optima in the reverse direction were 9.0 and 10.5 A role for this enzyme in T. gallinae metabolism is discussed.

Aldosterone biosynthesis and presence of cytochrome p-450 in the adrenocortical tissue of the chick embryo

By in vitro studies on the transformation of radioactive steroid substrates by adrenocortical preparations we have demonstrated the presence of the steroid-11 β-hydroxylase, 3 β-hydroxy-steroid: NAD(P) oxidoreductase (EC.1.1.1.51) and 3-oxosteroid Δ 4-Δ 5-isomerase (E5.3.3.1) systems in tissues of 10-day old embryos. In addition, the steroid-18-oxygenase, steroid-21-hydroxylase and the cholesterol side chain cleavage system were found to be present in adrenocortical tissues from day 14 of incubation onwards. About I picomole of cytochrome P-450 was detected per chick adrenal at day 12 of incubation and the level of this component increased rapidly after day 15. At the age of 12 days the serum Na + and K 4 concentrations measured by flame photometry were found to be 130.5 and 3.5 mEq/1 respectively whereas in the whole egg the concentrations were 48.8 mEq/1 for Na + and 28.5 mEq/1 for K +. It is concluded that aldosterone can be synthetized in adrenocortical tissue of chick embryos from day 14 of embryogenesis and therefore it may already play an important role in the regulation of plasma electrolytes.

Saccharopine Dehydrogenase: A KINETIC STUDY OF COENZYME BINDING

In addition to a high degree of specificity toward its amino acid and keto acid substrates, saccharopine dehydrogenase (e-N-( l -glutaryl-2)- l -lysine: NAD oxidoreductase (lysine-forming)) showed a strict coenzyme specificity. NADP+ did not support the reaction in the direction of oxidative cleavage of saccharopine, and NADPH was a poor coenzyme in the reverse direction. NADPH was bound by the enzyme with much less affinity than NADH, and its binding resulted in a large increase in Michaelis constants for substrates which add subsequently. Examination with the compounds that are fragments of the coenzyme (NAD+ or NADH) molecule revealed that adenosine was the smallest fragment that inhibited the reaction. The presence of a phosphate or pyrophosphate group at the 5′ position of the adenosine moiety greatly increased, while the presence of phosphate at or deletion of the oxygen from the 2′ position decreased the inhibitory action. NMN+, NMNH, guanine and hypoxanthine nucleotides, and pyrimidine nucleotides were not inhibitory. Kinetic analyses of the inhibition by AMP, ADP, ADP ribose, and ATP showed that they were competitive with respect to the coenzyme, and noncompetitive with respect to the other substrates, and indicate that they are bound at the coenzyme-binding site. The apparent inhibition constants were not altered by the variation in the concentrations of the second substrates, suggesting that the binding of these nucleotides was not followed by the combination of substrates.

Binding studies with bovine liver udp- d-glucose dehydrogenase

The direct binding of uridine 5′-(α- d-glucopyranosyluronic acid pyrophosphate) (UDP- d-glucuronic acid) and of uridine 5′-(α- d-xylopyranosyl pyrophosphate) (UDP- d-xylose) to bovine liver UDP- d-glucose:NAD oxidoreductase (EC 1.1.1.22) has been measured by equilibrium dialysis. At saturation, the hexameric enzyme binds six molecules of UDP- d-glucuronic acid to noninteracting sites. UDP- d-xylose binds to 2 distinct classes of sites, each class binding six molecules of ligand. UDP- d-xylose is able to displace either UDP- d-glucose or UDP- d-glucuronic acid and UDP- d-glucose is able to displace UDP- d-glucuronic acid from the enzyme. It is proposed that the enzyme displays half-of-the-sites reactivity toward both substrate (UDP- d-glucose) and cosubstrate (NAD) and all-of-the-sites reactivity toward UDP- d-glucuronic acid. UDP- d-xylose is considered to bind cooperatively with high affinity to six regulatory sites and independently with lower affinity to six catalytic sites on the enzyme. Active-enzyme centrifugation studies show that UDP- d-glucose: NAD oxidoreductase is hexameric at concentrations corresponding to those used in steady-state kinetic measurements.

Room temperature phosphorescence and the dynamic aspects of protein structure.

While the phosphorescence of aromatic chromophores in solution is normally quenched through diffusion of dissolved oxygen and other solvent-mediated processes, the phosphorescence of some proteins in solution is observed at room temperature. The tryptophan phosphorescence arises from residues which are hindered from interaction with oxygen by the folding of the polypeptide chains. Measurements of the phosphorescence lifetime of horse liver alcohol dehydrogenase (alcohol: NAD(+) oxidoreductase, EC 1.1.1.1) as a function of oxygen concentration indicate that internal tryptophan residues are periodically exposed to oxygen. This permits the calculation of rate constants for conformational oscillations in the enzyme. The present article illustrates the feasibility of employing phosphorescence in the study of proteins in solution in general and the utility of such experiments in probing the dynamic aspects of protein structure.

The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia.

1. The cytosol alcohol dehydrogenase (alcohol-NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO(2) (95:5) or of O(2)+CO(2) (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO(2) (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ;ADHI-air'. 4. In addition to fraction ADHII, cells grown in the presence of O(2) have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ;ADHI-O(2)', account for about 10% of the total activity. 5. The ADHI fractions (air) and (O(2)) have similar broad pH-activity curves and similar kinetic properties, both having a lower K(m) for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

Properties of the nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase from Blastocladiella emersonii

The regulatory properties of the NAD-specific isocitrate dehydrogenase ( threo- d s-isocitrate:NAD oxidoreductase (decarboxylating), EC 1.1.1.41) from Blastocladiella emersonii have been studied with steady state kinetic methods. The enzyme was found to catalyze the oxidative decarboxylation of isocitrate as well as the reversed reaction. The results show that the enzyme is extremely sensitive to the buffer concentration in the assay mixture. This inhibition is reversed by the activators, AMP and citrate and is competitive with regard to isocitrate. AMP reduces K m for the substrates but has no effect on V. The enzyme exhibits positive homotropic cooperativity towards isocitrate both in the absence and presence of activators, while the cooperativity towards NAD disappears in the presence of activators as well as high isocitrate concentrations. The activation by AMP occurs according to normal Michaelis Menten kinetics. AMP increases the pH optimum of the reaction. The experimental results are consistent with the possibility that the enzyme contains two binding sites for isocitrate, a catalytic and a regulatory site. Binding of citrate as well as AMP mimics the effect of binding of isocitrate to the regulatory site.

Glutamate dehydrogenase of Tetrahymena

Glutamate dehydrogenase [ l-glutamate: NAD(P) oxidoreductase (deaminating), EC 1.4.1.3] located in the mitochondria and able to utilize NAD, NADP, NADH or NADPH as substrate, has been purified 67-fold from Tetrahymena pyriformis. The activity with the four pyridine nucleotide substrates was catalyzed by a single enzyme as indicated by the constant association of the activities during purification and the coincidence of migration in polyacrylamide gel electrophoresis. Activity with NAD or NADH was stimulated by ADP and inhibited by GTP, Zn 2+, α-ketoglutarate, NADPH and NADH. NADH oxidation was inhibited by NH 4Cl or NaCl. Stimulation with ADP was strongly pH-dependent. Activity with NADPH was unaffected by ADP, GTP or Zn 2+ but was inhibited by NADPH and high concentrations of NH 4Cl or Nacl. The NADH- or NADPH-utilizing activity per mg protein was unaffected by a treatment of cells with chloramphenicol which caused a decrease in the specific activity of succinate dehydrogenase. The specific activity of NADH- and NADPH-utilizing activities increased coordinately by a factor of 2.7 during culture growth.

SOME ENZYMIC ASPECTS OF THE PRODUCTION OF OXIDIZED OR REDUCED METABOLITES OF CATECHOLAMINES AND 5‐HYDROXYTRYPTAMINE BY BRAIN TISSUES

AbstractThe Michaelis constants of purified aldehyde dehydrogenase (aldehyde: NAD oxidoreductase, EC 1.2.1.3) and aldehyde reductases (alcohol: NADP oxidoreductase, EC 1.1.1.2) from pig brain have been obtained for a number of biologically important aldehydes. The aldehydes include 3,4‐dihydroxyphenylacetaldehyde, D‐3,4‐dihydroxyphenylglycolaldehyde, and 5‐hydroxyindoleacetaldehyde. The relative activities of the aldehyde‐catabolizing enzymes in the soluble fractions of the cerebral cortex and caudate nucleus of pig brain have also been obtained. The values are used to show that the metabolic fates of the various aldehydes—and hence of the parent amines—may be explained in terms of the simple kinetics of these enzymes. It is also shown that the metabolic fates of the aldehydes may be influenced by their rates of synthesis. As the rate of aldehyde production increases the proportion of aldehyde reduced may be expected to increase at the expense of the proportion of aldehyde oxidized.It is further concluded from the kinetic constants that selective inhibition of aldehyde dehydrogenase may greatly affect the catabolism of dopamine and 5‐hydroxytryptamine by altering the relevant aldehyde concentrations, while the catabolism of norepinephrine is little affected under these circumstances. Conversely, it is concluded that selective inhibition of the aldehyde reductases should scarcely affect the catabolism of dopamine and 5‐hydroxytryptamine, but that the catabolism of norepinephrine should be markedly affected.The results also indicate that the concentrations of the various deaminated metabolites of the biogenic amines could be selectively controlled by modulation of the activity of the enzymes of aldehyde catabolism in brain.

Enzymic method for the quantitative determination of reduced glutathione

A new specific and sensitive assay method for reduced glutathione has been developed. It is based on the reaction HCHO + NAD + + H 2O → HCOOH + NADH + H +, catalyzed by formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC.1.2.1.1) in the presence of reduced glutathione. Oxidized glutathione and other thiols do not interfere in this reaction. A purification procedure for formaldehyde dehydrogenase from beef liver is presented. The influence of cysteine and some other thiols, leucine, ascorbate, lactate, pyruvate and four acids generally used for deproteinization is reported. The results obtained by this method from human blood and rat tissues are compared with those obtained by Ellman's method.

Assay of Aspartate Aminotransferase Activity: Effects of Serum and Serum Proteins on Oxalacetate Decarboxylation and Dialysis

Abstract Effects of serum and protein on assays measuring aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) were examined. Increasing amounts of normal human serum or human serum albumin in samples with fixed amounts of purified enzyme decrease absorbance values and thus distort the results obtained in an assay in which diazonium-salt chromogenic reagent is used. Serum also directly accelerates disappearance of oxalacetate from an incubation medium similar to that of the enzyme assay studied. Protein accelerates spontaneous decarboxylation of oxalacetate to pyruvate, preventing its quantitation in assays specific for oxalacetate. Primary amine groups of protein are evidently involved, because acylated albumin does not accelerate decarboxylation. This effect is not attributable to metal ions in serum and is not significant in procedures involving use of the less specific dinitrophenylhydrazine, which measure the decarboxylation product, pyruvate. Continuous kinetic assays of aspartate aminotransferase activity, in which oxalacetate is not allowed to accumulate, are unaffected by protein concentration. Fivefold dilution of patients’ sera with physiological saline results in activities that are artifactually high by 56% in affected assays. Varying oxalacetate decarboxylation is also a source of error in the estimation of malate dehydrogenase (L-malate:NAD oxidoreductase; EC 1.1.1.37) activity in body fluids. Protein increases the values obtained by automated procedures requiring dialysis by increasing the amount of oxalacetate diffusing into the chromogenic flow stream. A solution of albumin, about 6 g/dl, is suggested as a dilution fluid for use with the affected assays.

The purification and properties of an alcohol dehydrogenase from mouse liver

1. 1. An alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC ( i · i · i) was isolated from the supernatant fraction of mouse liver and purified 206-fold. 2. 2. A number of aliphatic aldehydes, ranging from acetaldehyde to octadecanal, as well as benzaldehyde and isobutanal, were capable of being reduced by the enzyme. K m values ranged from 9.9 × 10 −4 to 2.5 × 10 −6 M. 3. 3. Cetyl alcohol was slowly oxidized to the aldehyde by the enzyme m the presence of NAD +. However, attempts to oxidize ethanol were unsuccessful. 4. 4. The enzyme showed a pH optimum at pH 6.8 with irreversible denaturation occurring below pH 5 and above pH 9.

Biosynthesis of the Monoguanidinated Inositol Moiety of Bluensomycin, a Possible Evolutionary Precursor of Streptomycin

Abstract The general pattern of biosynthesis of the bluensidine (1D-1-O-carbamoyl-3-guanidino-3-deoxy-scyllo-inositol) moiety of bluensomycin, a monoguanidinated analogue of dihydrostreptomycin, has been studied in extracts of Streptomyces hygroscopicus forma glebosus ATCC 14607 (S. glebosus). Our results are consistent with the following biosynthetic pathway: myo-inositol (C)/→ keto-scyllo-inositol (D)/→ aminodeoxy-scyllo-inositol (E)/→ 1-amino-1-deoxy-scyllo-inositol-4-P (U)/→ 1D-1-O-carbamoyl-3-amino-3-deoxy-scyllo-inositol-6-P (F)/→ 1D-1-O-carbamoyl-3-guanidino-3-deoxy-scyllo-inositol-6-P →→ bluensomycin. A major uncertainty concerns the step (U) at which the carbamoyl group is introduced. Carbamoylation might occur, for example, prior to phosphorylation or following transamidination. myo-Inositol:NAD oxidoreductase activity (C) is reported for the first time in extracts of Streptomyces; activity was assayed by two radiochemical methods, both involving coupling with aminotransferase (D). l-Glutamine:keto-scyllo-inositol aminotransferase (D) was also found to catalyze aminodeoxy-scyllo-inositol:keto-scyllo-inositol, aminodeoxy-scyllo-inositol:pyruvate, and 1,3-diamino-1,3-dideoxy-scyllo-inositol:keto-scyllo-inositol transaminations. l-Arginine:inosamine-P amidinotransferase (F) catalyzed transamidinations with the following compounds as amidino acceptors: NH2OH, 1-amino-1-deoxy-scyllo-inositol-4-P, 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol-6-P, 1D-1,3-diamino-1,3-dideoxy-scyllo-inositol-6-P, 1D-1,3-diamino-1,2,3-trideoxy-scyllo-inositol-6-P, and a compound present in S. glebosus extracts believed to be 1D-1-O-carbamoyl-3-amino-3-deoxy-scyllo-inositol-6-P. The latter transamidination is markedly enhanced in crude extracts by carbamoyl-P. S. glebosus extracts also have 1-guanidino-1-deoxy-scyllo-inositol-4-P phosphohydrolase activity; neither this enzyme nor the corresponding enzyme from streptomycin producers can dephosphorylate the transamidination product presumed to be bluensidine-6-P. Acid hydrolysis of the latter compound gave a compound which, unlike the unhydrolyzed compound, was converted to 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol by enzymes from a streptomycin producing strain. S. glebosus cannot carry out the above conversion since it apparently lacks at least two enzymes which occur in streptomycin producers: guanidinodeoxy-(scyllo)-inositol dehydrogenase and l-alanine:1D-1-guanidino-3-keto-1-deoxy-scyllo-inositol aminotransferase. It is suggested that streptomycin producing strains might be descendents of an ancestral strain which, like S. glebosus, produced the monoguanidinated inositol derivative, bluensomycin. It is further suggested that gene duplication and subsequent evolutionary divergence resulted in biosynthesis of the diguanidinated inositol derivative, streptomycin, which is 10 times more effective than bluensomycin as an antibiotic and inhibitor of protein biosynthesis.

The blocking of the tetrachloroplatinate (II) inhibition of malate dehydrogenase by sulfur-containing amino acids

The inhibition of malate dehydrogenase ( l-malate:NAD oxidoreductase, EC 1.1.1.37) by the tetrachloroplatinate (II) complex, PtCl 4 2−, in the presence of various concentrations of the amino acids d l-methionine and l-cysteine was measured. The relative concentrations of PtCl 4 2− to malate dehydrogenase was 100:1. From the data, the half-life and the relative rate of inhibition in the presence and absence of the amino acids were calculated. Using these values it was possible to calculate a stability constant for each system. The stability constants for the malate dehydrogenase-PtCl 4 2−- l-cysteine ( K c ), malate dehydrogenase-PtCl 4 2−- d l-methionine ( K m ) and, malate dehydrogenase-PtCl 4 2−( K E ) were 56 M −1, 917 M −1 and 8·10 5 M −1, respectively. The reversibility of the malate dehydrogenase-PtCl 4 2− complex was also demonstrated, by addition of d l-methionine to the completely inhibited enzyme. About 40% of the enzyme activity was regenerated. Using the stability constants it was calculated that 27% of the enzyme's activity should be regenerated. From the results it is suggested that the free platinum complexes may be maintained in solution for a longer period of time if some or all of the halide ligands were replaced by sulfur groups from molecules like cysteine or methionine.

Stereochemistry of hydrogen transfer between pyridine nucleotide and some intermediates of cholesterol catabolism catalyzed by liver alcohol and aldehyde dehydrogenase

The stereochemistry of hydrogen transfer between pyridine nucleotide and some intermediates of cholesterol catabolism was studied. 1. 1. The hydrogen transferred from NADH to 3α,7α,12α-trihydroxy-5β-cholestan-26-al, 3-oxo-5β-cholan-24-oic acid and to acetaldehyde catalyzed by rat liver alcohol:NAD oxidoreductase (EC 1.1.1.1) was the 4A-hydrogen. 2. 2. The hydrogen transferred from 3α,7α,12α-trihydroxy-5β-cholestan-26-al and from acetaldehyde to NAD + catalyzed by horse liver aldehyde dehydrogenase (EC 1.2.1.3) was the 4A-hydrogen.

Rabbit muscle l-glycerol-3-phoshate dehydrogenase : Substrate activity of 3,4-dihydroxybutyl 1-phosphonate and 4-hydroxy-3-oxobutyl 1-phosphonate

The phosphonic acid analogs of glycerol 3-phosphate, 2,3-dihydroxypropyl 1-phosphonate and 3,4-dihydroxybutyl 1-phosphonate, were examined as substrates for the rabbit muscle NAD-linked glycerol-3-phosphate dehydrogenase ( l-glycerol-3-phosphate: NAD oxidoreductase, EC 1.1.1.8). The three-carbon analog was completely inert while the four-carbon analog was oxidized at approximately the same rate and had nearly the same K m (240 μM for glycerol 3-phosphate compared to 190 μM for 3,4-dihydroxybutyl 1-phosphonate) as the natural substrate. The rate of reduction of dihydroxyacetone phosphate was approx. 25 times faster than that for its analog, 4-hydroxy-3-oxobutyl 1-phosphonate. This difference was not due to a difference in K m values since the value for the natural substrate is 130 μM compared to 182 μM for the analog. The difference in rate does not appear to be due to a difference in the acidity of the phosphonate as compared to the natural substrate.

The quality control of serum lactate dehydrogenase isoenzyme separations using commercial quality control sera

The lactate dehydrogenase ( l-lactate:NAD oxidoreductase, EC 1.1. 1.27; LDH) isoenzyme content of 31 commercially available quality control sera was determined using thin layer agarose and fluorimetry. Only one of these sera was specifically intended for isoenzyme quality control purposes. The group of human sera, with no additives, proved to be the only one having an LDH isoenzyme distribution suitable for quality control use. Within-batch and between-batch precision has been determined over a 2–3 month period for LDH isoenzyme separations using several human quality control sera. The control of quality of this procedure has been shown to be feasible.