Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.     

Purification and properties of alanine dehydrogenase from Bacillus sphaericus.

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.     

Studies of lactate dehydrogenase in the purified state and in intact erythrocytes.

Lactate dehydrogenase in intact erythrocytes was studied by observing isotope exchange between lactate and pyruvate by p.m.r. The inhibition of the enzyme in intact cells by both oxalate and pyruvate was found to be similar to that of the purified enzyme. The activity of the enzyme in intact cells indicates that the free solution NAD+ + NADH concentration in erythrocytes is about 10 microM whereas the total extractable NAD+ + NADH is about 80 nmol/ml of cell water.     

Specific binding of cyclic ADP-ribose to calcium-storing microsomes from sea urchin eggs.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ which is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that it may be a general messenger for Ca2+ mobilization in cells. In this study I address questions of whether an intracellular receptor for cADPR exists and, if so, whether it is different from the IP3 receptor. A procedure employing nitrogen decompression was used to homogenize sea urchin eggs, and the Ca2(+)-storing microsomes were separated from mitochondria and other organelles by Percoll density centrifugation. Radioactive cADPR with high specific activity was produced by incubating [32P]NAD+ with the synthesizing enzyme and the product purified by high pressure liquid chromatography. The enzyme was membrane bound and was isolated from dog brain extracts by sucrose density gradient centrifugation. Partial purification of the enzyme was achieved by DEAE ion-exchange chromatography after solubilization with 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate. Specific binding of 32P-labeled cADPR to a saturable site on the Ca2(+)-storing microsomes was detected by a filtration assay. Scatchard analysis indicated a binding affinity of about 17 nM and a capacity of about 25 fmol/mg protein. The binding was not affected by either NAD+ (the precursor) or ADP-ribose (the hydrolysis product) at 0.5 microM but was eliminated by 0.3 microM nonlabeled cADPR. The receptor for cADPR appeared to be different from that of IP3 since IP3 was not an effective competitor at a concentration as high as 3 microM. Similarly, heparin at a concentration that inhibits most of the IP3-induced calcium release from the microsomes did not affect the binding. The binding showed a prominent pH optimum at about 6.7. Calcium at 40 microM decreased the binding by about 50%. These dependencies of the binding on pH and Ca2+ are different from those reported for the IP3 receptor and provide further support that the intracellular receptors for cADPR and IP3 are different.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation, purification, and some properties of NAD-dependent isocitrate dehydrogenase from the organic acid-producing yeast Yarrowia lipolytica

The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.     

Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.     

A bioluminescence method of determining the activity of NAD-dependent hydrogenase]

An analytical multienzyme system composed of NAD-dependent hydrogenase of Alcaligenes eutrophus, and reductase and luciferase from luminous bacteria was studied. The rate of luminescence increase of this system was found to be proportional to hydrogenase activity. The apparent Michaelis constants for NAD and hydrogen were determined (5 and 40 microM, respectively). The pH optimum is 7.5-9.0. Over the NAD concentration range from 20 to 100 microM, the rate of luminescence increase changed by less than 10%. At higher concentrations of NAD a monotonous decreasing of the rate of luminescence increase was observed. The proposed multienzyme system can be used for measuring the hydrogenase activity and hydrogen concentration. The high sensitivity to hydrogen (0.1 nmol in sample) and to hydrogenase (0.5 mU) and specificity of the system enable its application in the development of a biosensor for rapid detection of hydrogen in a medium.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

Dinucleosidetriphosphatase from rat brain

Rat brain P1,P3-bis(5'-adenosyl)-triphosphate adenylohydrolase (dinucleosidetriphosphatase, EC 3.6.1.29) was purified 1000-fold. The enzyme hydrolyzed diadenosine and diguanosine triphosphates (Km values 14 and 40 microM, and relative V 100 and 40, respectively) to the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was hydrolyzed at a residual rate. Diadenosine di and tetraphosphates, NAD+, and artificial phosphodiesterase substrates were not hydrolyzed. Bivalent cations [Mg(II), Mn(II) or Ca(II)] were required for activity, but Zn(II) was a competitive inhibitor (Ki = 5 microM). The optimum pH value was about 7.5. A molecular mass of 34 kdalton (gel filtration) and an isoelectric point of 5.5 (chromatofocusing) were found.     

NAD+ Glycohydrolase of the Plasma Membrane Prepared from Glial and Neuronal Cells

NAD+ glycohydrolase (EC 3.2.2.5) activity was detected in the plasma membrane prepared from the primary culture of rat astrocytes. The enzyme has a broad optimum pH range. From the kinetic analysis, a Michaelis constant of 91.2 microM and a maximum velocity of 0.785 mumol/min/mg protein were obtained. ADPribose exhibited a competitive inhibition with respect to NAD. The inhibition by nicotinamide was shown to be of a non-competitive type. ATP and GTP were found to be competitive inhibitors. NAD+ glycohydrolase activity was not detected in the plasma membrane prepared from the primary culture of neuronal cells of chick embryos.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat.

1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2--1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.     

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.     

Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate

Inositol trisphosphate (IP3) was previously shown to release Ca2+ from a nonmitochondrial store in sea urchin eggs. In this study, egg homogenates and purified microsomes were monitored with either fura 2 or Ca2+-sensitive minielectrodes to determine whether other stimuli would induce Ca2+ release. Pyridine nucleotides (whose concentrations are known to change at fertilization) were found to release nearly as much Ca2+ as did IP3. Average releases/ml of homogenate were: 0.6 microM IP3, 10.9 nmol of Ca2+; 50 microM NADP, 7.3 nmol of Ca2+; and 100 microM NAD, 6.5 nmol of Ca2+ (n = 6). Specificity was demonstrated by screening a series of other phosphorylated metabolites, and none was found to reproducibly release Ca2+. Calcium release induced by IP3 or NADP was immediate, whereas a lag of 1-4 min occurred with NAD. This lag before NAD-induced Ca2+ release led to the discovery that a soluble egg factor (Mr greater than 100,000) converts NAD into a highly active metabolite that releases Ca2+ without a lag. The NAD metabolite (E-NAD) was purified to homogeneity by high pressure liquid chromatography and produced half-maximal Ca2+ release at about 40 nM. Injection of E-NAD into intact eggs produced both an increase in intracellular Ca2+ (as assayed with indo-1) and a cortical reaction. Following Ca2+ release by each of the active agents (IP3, NAD, and NADP), the homogenates resequestered the released Ca2+ but were desensitized to further addition of the same agent. A series of desensitization experiments showed that homogenates desensitized to any two of these agents still responded to the third, indicating the presence of three independent Ca2+ release mechanisms. This is further supported by experiments using Percoll density gradient centrifugation in which NADP-sensitive microsomes were partially separated from those sensitive to IP3 and NAD.     

Complex formation between nucleotides and D-beta-hydroxybutyrate dehydrogenase studied by fluorescence and EPR spectroscopy

D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-requiring enzyme which specifically requires phosphosphatidylcholine for enzymic activity. The phosphatidylcholine modifies the binding and orientation of the coenzyme, NAD(H), with respect to the enzyme. In the present study, two derivatives of NAD, spin-labeled either at N-6 or C-8 of the adenine ring, were found to be active as coenzyme. The binding affinity of NADH to the enzyme was opitimized by increasing the salt concentration and increasing the pH from 6 to 8, with the pK at 6.8. Monomethylmalonate, a substrate analogue, was found to enhance NADH binding (Kd is reduced from 4 to 1 microM). Sulfite strongly enhances the binding of NAD+ via the enzyme-catalyzed formation of an adduct of sulfite with the nucleotide; the Kd for binding of NAD-sulfite is in the micromolar range, whereas NAD+ binding is more than a magnitude weaker. The binding of spin-labeled NAD(H) was further characterized by EPR spectroscopy. Increased sensitivity and resolution were obtained with the use of NAD(H) analogues perdeuterated in the spin-label moiety. For these analogues bound to D-beta-hydroxybutyrate dehydrogenase in phospholipid vesicles, EPR studies showed the spin-label moiety to be constrained and revealed two distinct components. Increasing the viscosity of the medium by addition of glycerol affected the EPR spectral characteristics of only the component with the smaller resolved averaged hyperfine splitting. The stage is now set to study motional characteristics of the enzyme, using these spin-labeled probes which mimic the coenzyme.     

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.     

ADP-ribosylation of platelet actin by botulinum C2 toxin

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively. The Km value of the ADP-ribosylation reaction for NAD was about 1 microM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively. ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.     

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

Effect of phenobarbital and 3-methylcholanthrene on aldehyde dehydrogenase activity in cultures of HepG2 cells and normal human hepatocytes

Aldehyde dehydrogenase (ALDH) activity was measured in primary cultures of normal human hepatocytes and of the human hepatoma cell line HepG2 after application of phenobarbital (PB) or 3-methylcholanthrene (MC) for 5 days. Treatment with PB alone resulted in a significant increase in both protein and DNA content at concentrations of 2 and 3 mM. Treatment with MC at a concentration as low as 5 microM led to a significant loss of cells when it lasted more than 5 days. Concentrations of 3-5 mM of PB in the media of HepG2 cell cultures caused a 2-fold enhancement of the activity of ALDH, as measured with NAD and propionaldehyde (P/NAD) or benzaldehyde (B/NAD). On the other hand, MC-treated cultures (5 microM) showed a 20-fold increase in enzyme activity measured with NADP and benzaldehyde (B/NADP), and a 2-fold increase in B/NAD activity. Combined treatment with both PB and MC led to an effect of dynamic synergism as far as B/NAD and B/NADP activities are concerned, suggesting a metabolite of MC as the mediator for the increase of ALDH activity. Normal human hepatocytes in primary cultures responded to PB (3 mM) in a similar way as HepG2 cells as far as DNA and protein content and ALDH activity are concerned. It is concluded, that HepG2 hepatoma cells behave similar to the normal hepatocytes in terms of ALDH regulation and can be used for studies on the activity of ALDH as modified by added xenobiotics.     

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.