mM For NAD Research Articles

A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus-transformed and non-transformed BHK-21/C13 fibroblasts

We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein-ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6–18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD + ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10–150 μg led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 μg of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis of 32P-labelled proteins. A variety of proteins are modified and their molecular weights depend on the NAD concentration in the permeabilized cell incubation. At 0.5 μM NAD + there were two major acceptors with M r values of 14 kDa and 30 kDa, and at 100 μM NAD +, three major acceptors, with M r values of 19 kDa, 45 kDa and greater than 170 kDa. NAD concentrations of between 1 μM and 100 μM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of M r > 170 kDa, pointing to a critical difference around 0.5–1.0 μM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents. Since the molecular mass values were less than 12 kDA, we presume the proteins (or protein) are non-histones. Kinetics of NAD + utilization show an initial burst of activity. Autoradiographs show little further change in protein labelling pattern or intensities after the first 10 s of an incubation. The overall rate of ADPribose degradation is slow in these permeabilized cells, only about 30–35% being lost in a 90 min degradation assay. Differences are seen in the autoradiographs, however, between the two cell types when 32P-labelled proteins, solubilized at different times during the degradation assay, are electrophoresed in the polyacrylamide gels. Finally, 3-aminobenzamide takes about 15 min to completely inhibit ADPribose synthesis in the permeabilized cells. During this time, synthesis is seen from autoradiographs to be limited to the modification of three particular proteins of molecular mass 25 kDa, 54 kDa and 170 kDa.

Cellular regulation of poly(ADP) ribosylation of proteins: I. Comparison of hepatocytes, cultured cells and liver nuclei and the influence of varying concentrations of NAD

The in vitro rates ( v init) of poly(ADP-ribose) polymerase of permeabilized rat hepatocytes and of nuclei, isolated from hepatocytes, did not differ significantly. Incubation beyond 3 min resulted in diminished poly(ADP) ribosylation in hepatocytes compared with nuclei, coinciding with high rates of plasma membrane-associated NAD-glycohydrolase. Cultured cells ( Drosophila K c cells, gliosarcoma 9L, human fibroblasts and mouse spleen lymphocytes) exhibit variations of NAD-glycohydrolase and poly(ADP-ribose) polymerase activities and the assessment of poly(ADP-ribose) polymerase activity in permeabilized cells requires simultaneous assay of NAD-glycohydrolase. In rat liver nuclei during 10 min incubation with 500 μM NAD, 40% of NAD is consumed, 10% ADP-ribose is bound to proteins, and 20% ADP-ribose, 5% AMP and 2.7% adenosine are liberated. As determined by solvent partitioning (Jackowski, G & Kun E, J biol chem 258 (1983) 12587) [1], the phenol-soluble protein-ADP-ribose fraction represents largely mono(ADP)-ribose protein adducts, whereas the H 2O-soluble phase contains poly(ADP)-ribosylated proteins. The quantity of ADP-ribose protein adducts, the chain length of oligomers and the nature of apparent acceptor proteins in liver nuclei vary significantly with the concentration of NAD as substrate. At 500 μM NAD concentration the quantity of ADP-ribose containing adducts was in the nmol per mg DNA range, the polymers are long chains and the acceptor proteins predominantly non-histone proteins. At 0.1 μM NAD as substrate pmol quantities of monomeric ADP-ribose adducts per mg DNA were formed and the main acceptors were sharply discernable on the basis of molecular mass as histones, high mobility non-histone proteins, two protein groups of a mass of 66 and 44 kD respectively, and the poly(ADP-ribose) polymerase enzyme protein of 119 kD mass. Whereas products in the presence of 0.1 μM NAD may indicate acceptors of highest reactivity, protein adducts formed in the presence of 500 μM NAD resemble a pattern found in vivo.

Purification and characterization of guinea pig liver morphine 6-dehydrogenase.

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone.

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD+ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD+ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of α-ketoisocaproate and α-ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD+ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.

Purification of arogenate dehydrogenase from Phenylobacterium immobile

Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The M r of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The K m values were 0.09 mM for arogenate and 0.02mM for NAD +. The enzyme has a high specificity with respect to its substrate arogenate.

Some characteristics of Δ1-pyrroline-5-carboxylate dehydrogenase in rat cerebellum

Δ 1-Pyrroline-5-carboxylate dehydrogenase (P5CDH) in rat cerebellum was assayed with a simple spectrophotometric method using high-speed supernatants of whole tissue homogenates. Kinetic analysis showed that the enzyme has K m values of 0.83 ± 0.21 mM for Δ 1-pyrroline-5-carboxylate and 0.47 ± 0.02 mM for NAD +. Various cations inhibited P5CDH but only at relatively high concentrations. Several amino acids were strongly inhibitory in the order GABA > glycine > hydroxyproline > cysteine ⋍ proline > glutamine > glutamate > alanine .

Some enzymatic properties of NAD +-dependent glutamate dehydrogenase of mussel hepatopancreas ( Mytilus edulis L.)—requirement of ADP

1. 1. The glutamate dehydrogenase of mussel hepatopancreas is NAD(H) dependent and shows absolute requirement for ADP as cofactor. 2. 2. The optimum pH for GDH lies between 7.4 and 8.2 (reductive amination) and 9.5 (oxidative deamination). The enzyme has an activation energy of 53.9 kJ/mol and an average Q 10 of 1.65. 3. 3. ADP and 2-oxoglutarate stabilize the enzyme, while NADH diminishes the stabilization significantly. 4. 4. In the sense of reductive amination the apparent K m values are 0.38 mM for 2-oxoglutarate, 34 μM for NADH and 20 mM for NH 4 +. In the opposite sense the apparent K m for glutamate is 5 mM and 0.55 mM for NAD +.

Arogenate dehydrogenase from Streptomyces phaeochromogenes. Purification and properties.

Arogenate dehydrogenase, the terminal enzyme of tyrosine biosynthesis in Streptomyces phaeochromogenes, was purified to homogeneity by a five-step procedure. The enzyme is a dimer of Mr 57 600 as determined by dodecyl sulfate polyacrylamide gel electrophoresis after cross-linking of the monomers, or of 66 300 as found by gel permeation chromatography, and consists of two identical subunits of Mr 28 100. The pI of the enzyme is 4.45, and the Km values are 0.105mM for arogenate and 0.01 mM for NAD.

Purification and some properties of UDP-N-acetylglucosamine 4-epimerase from Bacillus subtilis.

UDP-N-Acetylglucosamine 4-epimerase (EC 5.1.3.7) was purified from a cell extract of Bacillus subtilis by protamine sulfate treatment, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, and Sephadex G-150. The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 62,000 by gel filtration. The enzyme had an optimal pH in the range of 7.0 to 9.0, and was active at 30~35°C in the absence of added NAD and more active at 40~45°C in the presence of NAD. The enzyme activity was highly stimulated by small amounts of NAD. The estimated Km values were 4.4 mM for UDP-N-acetylglucosamine and 0.13mM for NAD. No UDP-glucose 4-epimerase activity was found in the purified enzyme. The enzyme was inhibited by high concentrations of glucosamine and inhibition by glucosamine was competitive with UDP-N-acetylglucosamine. The inhibition constant was 37.0 mM. N-Acetylglucosamine did not inhibit the enzyme.

Malate dehydrogenase from thermophilic Humicola lanuginosa and Mucor pusillus: purification and comparative properties of the enzymes with differing thermostabilities

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from the thermophiles Humicola lanuginosa and Mucor pusillus. The H. lanuginosa enzyme was homogeneous on sodium dodecyl sulphate – polyacrylamide gels, while the M. pusillus enzyme was more than 95% pure. The two enzymes appeared to be composed of two subunits of equal size, each of 36 000 daltons (H. lanuginosa) or 33 000 daltons (M. pusillus). The native enzymes revealed molecular weights of 68 000 as determined by gel filtration. The isoelectric points of malate dehydrogenase from H. lanuginosa and M. pusillus were 3.9 and 4.2, respectively. The reduction of oxaloacetate by the H. lanuginosa enzyme was optimum at pH 8.5–9 with apparent Km's of 0.12 mM for oxaloacetate and 0.027 mM for NADH. On the other hand, M. pusillus enzyme snowed a pH optimum of 7.8–8.5 with apparent Km's of 0.075 mM for oxaloacetate and 0.1 mM for NADH. The L-malate oxidation reaction was catalyzed optimally at pH 10 by the H. lanuginosa enzyme with apparent Km's of 5.8 mM for malate and 0.1 mM for NAD, while the M. pusillus enzyme catalyzed it optimally between pH 9.5 and 10 with apparent Km's of 4.44 mM for malate and 0.16 mM for NAD. The optimum temperature for reduction of oxaloacetate was 50 °C for both the enzymes. The H. lanuginosa enzyme was resistant to heat inactivation at 40 °C, but lost 60% of its activity after 15 min at 50 °C. Mucor pusillus enzyme, on the other hand, retained 90% activity at 60 °C after 10 min. The two enzymes were protected from heat inactivation by monovalent cations (viz Na+, K+, and NH4+), as well as citrate, which may possibly involve conformational changes.

NAD: Guanidino group specific mono ADP-ribosyltransferase activity in skeletal muscle

The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcolemma and sarcoplasmic reticulum from pig skeletal muscle contains NAD:dependent mono ADP-ribosyltransferase activity toward the guanidine analog, P- nitrobenzylidine aminoguanidine. No or little activity could be found in the sarcolemma or sarcoplasmic reticulum derived from canine cardiac muscle. Seventy percent of activity extracted from rabbit skeletal muscle is localized in the sarcoplasmic reticulum. The enzyme has a pH optimum of 7.4, and KM of 0.5 mM and 0.35 mM for NAD and p-nitro benzylidine aminoguanidine, respectively. Inorganic phosphate, KCl, and guanidine derivatives inhibit the reaction. Incubation of the sarcoplasmic reticulum or glycogen pellet with (adenylate-32P) NAD or [adenosine-14C(U)]-labeled NAD results in the incorporation of radioactivity into proteins. A large number of proteins are labeled in the sarcoplasmic reticulum fraction. The major labeled band in the glycogen pellet corresponds to a protein of molecular weight of 83 K.

Regulation of energy metabolism in Trypanosoma (schizotrypanum) cruzi epimastigotes. II. NAD +-dependent ǵlutamate dehydrogenase

Trypanosoma (Schizotrypanum) cruzi epimastigotes (EP stock) grown in complex LIT medium rapidly consume the glucose present but, under aerobic conditions, continue growth in its absence with the concomitant excretion of ammonia, suggesting the utilization of amino acids for energy production. A search for metabolic pathways responsible for amino acid oxidation led to the detection of a NAD +-dependent glutamate dehydrogenase ( l-glutamate: NAD + oxidoreductase, E.C. 1.4.1.2) which is different from an NADP +-dependent enzyme previously reported. The enzyme has been partially purified and its kinetic and regulatory properties studied in both directions of the reaction. K m values were 3.6 mM for α-ketoglutarate, 0.170 mM for NADH and 16 mM for NH + 4, V max = 0.67 μmol min −1/ mg −1 protein for aminative reduction; K m values were 23.5 mM for lglutamate and 2.9 mM for NAD +, V max = 0.02 μmol min −1 mg −1 protein for deaminative oxidation, Tris buffer, pH 7.6. The enzyme is strongly inhibited by ATP, GTP, ADP and GDP (50% inhibition at 0.75 mM ATP, 3 mM MgCl 2). S-Acetyl-CoA is also a potent inhibitor of the enzyme. The results demonstrate the presence of a specific pathway for the oxidation of amino acids, which is tightly regulated by the energy charge and the Krebs cycle activity in T. cruzi epimastigotes.

Malate dehydrogenase of Tetrahymena pyriformis: evolutionary and regulatory aspects

Abstract 1. 1. Tetrahymena pyriformis has two isoenzymes of malate dehydrogenase, I and II. After purifying (isoenzyme I, 85-fold), the mol. wt of each enzyme was determined to be around 122,000 with four subunits of 28,000. 2. 2. The Michaelis constants are: 0.25 mM for oxaloacetate (isoenzyme I), 0.8 mM for oxaloacetate (isoenzyme II), 4mM for malate (isoenzyme I and Il), 0.13 mM for NAD (isoenzyme I and II) and 0.24 mM for NADH (isoenzyme I and II). 3. 3. It was concluded that Tetrahymena malate dehydrogenase was closer to the enzyme from Gram positive bacteria than to that from eukaryotes.

NAD +-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD + hydrolysis

We previously demonstrated inhibition of Na +-dependent 32P i transport in canine renal brush-border membranes in association with NAD +-induced ADP ribosylation of membrane protein(s) and postulated that NAD + inhibits P i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD + resulted in release of P i which was prevented by EDTA. It was proposed that NAD +-mediated inhibition of 32P i transport might occur through this mechanism. To determine whether NAD + inhibited 32P i transport by a mechanism other than or in addition to release of P i, we compared Na +-dependent 32P i counterflow in brush-border membrane equilibrated with P i or with P i generated from NAD +. Release of P i from NAD + incubated with brush-border membrane was confirmed. The increased uptake of 32P i which was demonstrated in brush-border membrane equilibrated with P i was not measured when intravesicular P i was generated from a concentration of NAD + which effected ADP-ribosylation of brush border membranes (100 μM NAD +). In contrast, increased uptake of 32P i was demonstrated when intravesicular P i was generated from 1 μM NAD + which did not effect ADP ribosylation. Mg 2+-dependent ADP ribosylation of brush-border membrane incubated with NAD + was demonstrated which persisted during the time interval of 32P i uptake measurements. Our findings are compatible with the hypothesis that NAD +-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg 2+ and decreased ADP ribosylation.

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa.

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway has been purified to 96% homogeneity. The native enzyme, which appears to be a tetramer, has an apparent molecular weight of 210000. The purified enzyme shows a narrow pH optimum at pH 7.8 and does not require ions for its catalytic activity. Under standard assay conditions the enzyme acts preferentially with NAD but reduces NADP at 11% of the rate observed for NAD, primarily because of a difference in K m. Apparent K m values are 6.4 μM for 5-carboxymethyl-2-hydroxymuconic semialdehyde and 52.2 μM for NAD.

Spin echo proton NMR studies of the metabolism of malate and fumarate in human erythrocytes: Dependence on free NAD levels

The NAD-dependent conversion of malate to lactate in human erythrocytes was studied by spin echo proton NMR. A pathway involving the decarboxylation of oxaloacetate catalysed by haemoglobin is proposed to account for the observed reaction. NADP-dependent reaction was negligible. The rate of the reaction was measured in intact erythrocytes under controlled conditions. This rate correlates with that obtained with lysates at 30 μM free NAD and that obtained with purified human erythrocyte enzymes at about 15 μM NAD. The total extractable NAD in the intact cells was 70–90 μM. Experiments with cells containing elevated NAD levels could be explained by a significant fraction of the NAD being weakly bound ( K d about 1 mM) to haemogloin.

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis.

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus −30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q10 of 2.16. The Km for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The Km(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, Km's were 340 μM for glycerol-3-P and 12 μM for NAD+. Effects of most inhibitors (of the forward reaction), glycerol-3-P (Ki = 2.4 mM), NAD+ (Ki = 0.2 mM), ATP, Mg·ATP, and Pi, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the Vmax activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.

Effects of ethanol and other organic solvents on the kinetic behaviour of purified human placental oestradiol-17β-dehydrogenase

The effects of acetone, ethanol, dimethylsulphoxide, glycerol, and propanediol on the formation of reduced coenzyme in the presence of purified human placental oestradiol dehydrogenase were measured fluorometrically at 10 microM oestradiol-17 beta and 500 microM NAD. Addition of increasing amounts of each solvent except glycerol resulted in an initial increase followed by a decline in rate of the enzyme catalyzed reaction. Glycerol caused a linear decline in rate. A range (0.1-25%, v/v) of ethanol concentrations was tested for its effect upon the Km and Vmax values for oestradiol, NAD, and NADP. Over this range the Km value for NADP remained nearly constant (0.6-0.8 microM). The Km value for NAD increased rapidly with an upward convex shape of the curve (6.6-25 microM), and the Km value for oestradiol with NAD or NADP as cofactor increased slowly with an upward concave shape (6.7-25 microM). With increasing ethanol concentration, the maximum velocity increased, reaching its highest values at 10 and 20% ethanol for NAD and NADP, respectively, and then declined. At the maximum Vmax values for NAD and NADP were, respectively, 165 and 200% the values obtained with 0.1% ethanol.