Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine

Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2-trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5'-cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin.     

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.     

Molecular conversion of NAD kinase to NADH kinase through single amino acid residue substitution

NAD kinase phosphorylates NAD+ to form NADP+ and is strictly specific to NAD+, whereas NADH kinase phosphorylates both NAD+ and NADH, thereby showing relaxed substrate specificity. Based on their primary and tertiary structures, the difference in the substrate specificities between NAD and NADH kinases was proposed to be caused by one aligned residue: Gly or polar amino acid (Gln or Thr) in five NADH kinases and a charged amino acid (Arg) in two NAD kinases. The substitution of Arg with Gly in the two NAD kinases relaxed the substrate specificity (i.e. converted the NAD kinases to NADH kinases). The substitution of Arg in one NAD kinase with polar amino acids also relaxed the substrate specificity, whereas substitution with charged and hydrophobic amino acids did not show a similar result. In contrast, the substitution of Gly with Arg in one NADH kinase failed to convert it to NAD kinase. These results suggest that a charged or hydrophobic amino acid residue in the position of interest is crucial for strict specificity of NAD kinases to NAD+, whereas Gly or polar amino acid residue is not the sole determinant for the relaxed substrate specificity of NADH kinases. The significance of the conservation of the residue at the position in 207 NAD kinase homologues is also discussed.     

Inhibition and inactivation of bovine mammary and liver UDP-galactose-4-epimerases.

Bovine liver and mammary UDP-galactose-4-epimerases were investigated with respect to various inhibitors and inactivators. Uridine nucleotides and NADH are potent inhibitors with Ki values in the low micromolar range. The NAD+/NADH ratio may be an important physiological control mechanism for it affects markedly the activity of the enzyme with 50% inhibition occurring at a ratio of 20:1. In the presence of uridine nucleotides binding of NADH to the epimerases is enhanced. Consequently, the effect of changes in the NAD+/NADH ratio in vivo would not be immediately apparent as uridine nucleotides would slow down the displacement of NADH by NAD+. Neither uridine nor galactose 1-phosphate inhibits the purified enzymes as previously reported with the impure liver enzyme. Uridine nucleotides provide almost total protection against the apparent first order inactivation of the epimerases by trypsin and allow determination of dissociation constants. NAD+ partially protects against trypsin inactivation. Inactivation with various sulfhydryl reagents is complex and the results indicate that at least three sulfhydryl groups may be modified before total inactivation occurs. Partial inactivation occurs upon modification of the epimerases with 2-hydroxy-5-nitrogenzyl bromide. Some protection against this modification is provided by the combination of NAD+ and UDP.     

Characterization of NADH kinase from Saccharomyces cerevisiae

At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD+ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1,041-fold from the mitochondrial fraction. The Km values for NADH and ATP were 105 microM and 2.1 mM, respectively. The relative molecular mass was estimated to be 160,000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.     

Effects of nicotinamide coenzymes on the stability of enzyme activities and proteins in niacin-deficient quail tissues against trypsin treatment

The stability of liver and muscle enzymes and proteins in niacin-deficient quail towards trypsin treatment in the presence and absence of coenzymes, NAD or NADP, was characterized. The protection of liver dehydrogenases by coenzymes was low when they are subjected to trypsin digestion for 60 min. In contrast, in the muscle there was substantial protection against trypsin inactivation of glyceraldehyde-3-phosphate dehydrogenase by NAD and of 6-phosphogluconate dehydrogenase by NADP. Among all enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against trypsin inactivation by NAD. SDS-polyacrylamide gel electrophoresis demonstrated that muscle proteins from the niacin-deficient group were more substantially protected compared to control and pair-fed groups when liver and muscle extracts were spiked with NAD and subjected to trypsin digestion. Overall results suggest that niacin deficiency exerted specific destabilizing effects on the stability of enzymes and proteins in muscle.     

A Ca2+, Calmodulin-dependent NAD kinase from corn is located in the outer mitochondrial membrane

NAD kinase activity from dark grown corn coleoptiles is shown to be almost totally dependent on Ca2+ and calmodulin. Nearly all of the enzyme activity is found in a particulate fraction. Upon differential and density gradient centrifugation the NAD kinase activity co-migrates with the mitochondrial cytochrome c oxidase whereas marker activities for nuclei, etioplasts, endoplasmic reticulum, and microbodies could well be separated, indicating that the NAD kinase is associated with mitochondria. This NAD kinase, associated with intact mitochondria, can be activated by exogenously added Ca2+ and calmodulin. In order to investigate the submitochondrial localization of the NAD kinase, the organelles were ruptured by osmotic treatment and sonication and the submitochondrial fractions were separated by density gradient centrifugation. The NAD kinase activity exhibits the same density pattern as the antimycin A-insensitive NADH-dependent cytochrome c reductase, a marker enzyme of the outer mitochondrial membrane. Marker enzymes for the mitochondrial matrix and the inner mitochondrial membrane reveal different density profiles. These results indicate that the Ca2+, calmodulin-dependent NAD kinase from coleoptiles of dark grown corn seedlings is located at the outer mitochondrial membrane. The physiological relevance of the location and the Ca2+, calmodulin-dependence of the NAD kinase will be discussed.     

Effect of aluminium on the NAD+ kinase activity of Euglena gracilis grown heterotrophically

The effect of 2 mM AlCl3 on NAD+ kinase (E.C. 2.7.1.23) activity was studied using Euglena gracilis strain Z grown heterotrophically in darkness at pH 3.5 in the presence of lactate as sole carbon source. The Al-treatment slowed down the culture growth and suppressed the peak of NAD+ kinase activity, which characterizes the beginning of the exponential phase of growth of the control cell cultures. There are two possible explanations of the Al effect: it 1) either prevents the enzyme activation by the Ca-calmodulin (CaM) complex; or 2) suppresses the CaM-dependent NAD+ kinase form. In Euglena cells, a part of the NAD+ kinase activity is enhanced by EGTA and lowered by Ca2+: this peculiar NAD+ kinase activity is unaffected by the Al treatment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.     

Nicotinamide 3,N4-ethenocytosine dinucleotide, an analog of nicotinamide adenine dinucleotide. Synthesis and enzyme studies.

A structural analog of NAD+, NICOTINAMIDE 3,N-4ethenocytosine dinucleotide (epsilonNCD+), has been synthesized, characterized, and compared in activity with the natural coenzyme in several enzyme systems. The Vmax and apparent Km values were determined for NAD+, epsilonNCD+, and epsilonNAD+ (nicotinamide 1, N6-ethenoadenine dinucleotide) with yeast alcohol, horse liver alcohol, pig heart malate, beef liver glutamate, and rabbit muscle lactate and glyceraldehyde-3-phosphate dehydrogenases. The Vmax for epsilonNCD+ was as great or greater than that obtained for NAD+ with three of the enzymes, 60-80 per cent with two others, and 14 percent with one. EpsilonNCD+ was found to be more active than epsilonNAD+ with all six dehydrogenases. EpsilonNCD+ served as a substrate for Neurospora crassa tnadase, but could not be phosphorylated with pigeon liver NAD+ kinase. NAD+ pyrophosphorylase from pig liver was unable to catalyze the formation of epsilonNCD+ from the triphosphate derivative of epsilon-cytidine and nicotinamide mononucleotide, but was able to slowly catalyze the pyrolytic cleavage of epsilonNCD+. The coenzyme activity of epsilonNCD+ with dehydrogenases can be discussed in terms of the close spatial homology of epsilonNCD+ and NAD+, which may allow similar accommodations within the enzyme binding regions.     

Calmodulin and calmodulin-mediated processes in plants

Abstract. The Ca²⁺ -binding protein calmodulin is found in all plants investigated so far. The comparison of the biochemical and functional properties reveals that it is structurally conserved and functionally preserved throughout the plant and animal kingdom. Among the plant enzymes so far known to be dependent on the Ca²⁺ -calmodulin complex are NAD kinase(s), Ca²⁺ -transport ATPase, quinate: NAD⁺ oxidoreductase, soluble and membrane bound protein kinases, and H⁺ -transport ATPase. Calmodulin may play also an important role in the regulation of other cellular reactions, such as hormone-mediated processes, secretion of enzymes, and contractile mechanisms. On the basis of the NAD kinase and its regulation by light and Ca²⁺ -calmodulin, it is suggested that changes in the cellular, free Ca²⁺ concentration following stimulation may alter the metabolism of a plant cell. According to this suggestion free Ca²⁺ may act as a second messenger in plants much as it does in animal cells.     

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

NAD+-kinase from Neurospora crassa: isolation and properties

The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively.     

Mesocestoides lineatus: trypsin induced development to adult mediated by Ca2+ and protein kinase C

A mode of action of the inducible treatment with trypsin for the development of Mesocestoides lineatus tetrathyridium to adult was analyzed by administering various agents effective on Ca2+-dependent metabolic pathways in the cells: protein kinase C activators such as a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, and a tumor promoting phorbol, 12-O-tetra-decanoyl-phorbol-13-acetate, enhanced the trypsin induced developmental processes. On the contrary, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, cyclic adenosine 3',5'-monophosphate, and adenylate cyclase activators such as forskolin and cholera toxin, inhibited the triggering action of trypsin. Furthermore, a combined administration of Ca2+ ionophore (A23187) and the phorbol showed a similar effect with trypsin treatment, and sodium taurocholate acted as a potent enhancer like the activators of protein kinase C. These results strongly suggest that the initiation of development to adult in this cestode may be regulated synergistically by Ca2+ and protein kinase C, and that a bile acid may be involved in an activation mechanism of protein kinase C.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae

Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.     

Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step.     

The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex.

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.     

Pressure-adaptive differences in proteolytic inactivation of M4-lactate dehydrogenase homologues from marine fishes

The inactivation by hydrostatic pressure of muscle-type lactate dehydrogenase (M4-LDH, EC 1.1.1.27; L-lactate: NAD+ oxidoreductase) homologues from five shallow-living and six deep-living marine teleost fishes was compared. The pressures which inactivate these enzymes are much higher than the pressures experienced by any of the species. To determine whether hydrostatic pressure effects on protein aggregation state and conformation might influence proteolysis, the inactivation of LDH by the proteases, trypsin (EC 3.4.21.4) and subtilisin (EC 3.4.4.16) was determined at atmospheric pressure and 1,000 atm pressure. At 10 degrees C and atmospheric pressure, the enzymes of the shallow-living fishes are inactivated four times faster by trypsin and three times faster by subtilisin than are the homologues of the deep-living species. At 1,000 atm pressure, the homologues of shallow-occurring fishes were inactivated 28 to 64% more than predicted from the summed effects of denaturation by 1,000 atm pressure and tryptic inactivation at atmospheric pressure. In contrast, the homologues of the deep-sea species were inactivated by trypsin 0 to 21% more than expected. At 1,000 atm, inactivation by subtilisin increased to a similar degree for enzymes from both deep- and shallow-living species. However, at 1,000 atm, the M4-LDH homologues of the deep-sea species lost less activity (55.3%) than did the homologues of the shallow species (86.4%). In comparisons made at 200 atm, a pressure typical of the habitat of the deep-occurring species, tryptic inactivation of the LDH of the shallow-living Sebastes melanops was increased 14%. No pressure inactivation of the enzyme is evident at 200 atm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative modulation of Escherichia coli NAD kinase by NADPH and NADH.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.