Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Regio- and stereospecificity of homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons

The homogeneous 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase EC 1.3.1.20), Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). Examination of the substrate specificity of the purified dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons indicates that the enzyme will catalyze the NAD(P)-dependent oxidation of trans-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, 5-methylchrysene, and benzo[a]pyrene under physiological conditions. Comparison of the utilization ratios Vmax/Km indicates that benzenedihydrodiol and the trans-1,2- and trans-7,8-dihydrodiols of 5-methylchrysene were most efficiently oxidized by the purified dehydrogenase, followed by the trans-7,8-dihydrodiol of benzo[a]pyrene and the trans-1,2-dihydrodiols of phenanthrene, chrysene, and naphthalene. The purified enzyme appears to display rigid regio-selectivity, since it will readily oxidize non-K-region trans-dihydrodiols but will not oxidize the K-region trans-dihydrodiols of phenanthrene and benzo[a]pyrene. The stereochemical course of enzymatic dehydrogenation was investigated by circular dichroism spectrometry. For the trans-1,2-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, and 5-methylchrysene, the dehydrogenase preferentially oxidized the (+)-[S,S]-isomer. Apparent inversion of this stereochemical preference occurred with the trans-7,8-dihydrodiol of 5-methylchrysene, as the (-)-enantiomer was preferentially oxidized. No change in the sign of the Cotton Effect was observed following oxidation of the racemic trans-7,8-dihydrodiol of benzo[a]pyrene, suggesting that both stereoisomers of this compound were substrates. Large-scale incubation of the [3H]-(+/-)-trans-7,8-dihydrodiol of benzo[a]pyrene with the purified dehydrogenase resulted in greater than 90% utilization of this potent proximate carcinogen, suggesting that the enzyme utilizes both the (-)-[R,R] and the (+)-[S,S]-stereoisomers, which confirms the circular dichroism result. These data show that dihydrodiol dehydrogenase displays the appropriate regio- and stereospecificity to catalyze the oxidation of both the major and minor non-K-region trans-dihydrodiols that arise from the microsomal metabolism of benzo[a]pyrene in vivo.     

Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2'-Dithiodipyridine is an activator of the enzyme.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

Benzo[a]yrenedione/benzo[a]pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen.

The ability of the isomeric quinone metabolites of benzo[a]pyrene, benzo[a]pyrene-6,12-dione, benzo[a]pyrene-1,6-dione, and benzo[a]pyrene-3,6-dione to undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrenediols and intermediate semiquinone radicals has been characterized. Under anaerobic conditions, all three benzo[a]pyrenediones are easily reduced to benzo[a]pyrenediols, even by mild biological agents such as NAD(P)H, cysteamine, and glutathione. The benzo[a]pyrenediols, in turn, are very rapidly autoxidized to the benzo[a]pyrenediones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autoxidations, and other reactive reduced oxygen species, such as the superoxide and hydroxyl radicals, are probably formed transiently as well. The benzo[a]pyrenediol-benzo[a]pyrenedione interconversions proceed by one-electron steps; the corresponsing semiquinone radicals can be monitored by electron spin resonance spectroscopy as inter mediates during these reactions carried out at high pH. Benzo[a]pyrenediones induce DNA strand scission when incubated with bacteriophage T7 DNA. This damage is modified by conditions which indicate that reduced oxygen species propagate the free-radical reactions responsible for the strand scission. Benzo[a]pyrenediones are electron-acceptor substrates for NADH dehydrogenase from Clostridium kluyveri. Catalytic amounds of these benzo[a]pyrene metabolites, together with this respiratory enzyme function as cyclic oxidation-reduction couples which link NADH and molecular oxygen in the continuous production of hydrogen peroxide. These data, together with preliminary results with cells in culture, indicate that benzo[a]pyrenediones are potentially harmful metabolites of benzo[a]pyrene, acting by processes which lead to their regeneration rather than depletion; nucleic acid and call damage is probably produced by the reactive reduced oxygen species resulting from such regenerative oxidation-reduction cycles.     

Mycorrhization alleviates benzo[a]pyrene-induced oxidative stress in an in vitro chicory root model

Among chemicals that are widely spread both in terrestrial and aquatic ecosystems, benzo[a]pyrene is a major source of concern. However, little is known about its adverse effects on plants, as well as about the role of mycorrhization in protection of plant grown in benzo[a]pyrene-polluted conditions. Hence, to contribute to a better understanding of the adverse effects of polycyclic aromatic hydrocarbons on the partners of mycorrhizal symbiotic association, benzo[a]pyrene-induced oxidative stress was studied in transformed Cichorium intybus roots grown in vitro and colonized or not by Glomus intraradices. The arbuscular mycorrhizal fungus development (colonization, extraradical hyphae length, and spore formation) was significantly reduced in response to increasing concentrations of benzo[a]pyrene (35–280 μM). The higher length of arbuscular mycorrhizal roots, compared to non-arbuscular mycorrhizal roots following benzo[a]pyrene exposure, pointed out a lower toxicity of benzo[a]pyrene in arbuscular mycorrhizal roots, thereby suggesting protection of the roots by mycorrhization. Accordingly, in benzo[a]pyrene-exposed arbuscular mycorrhizal roots, statistically significant decreases were observed in malondialdehyde concentration and 8-hydroxy-2′-desoxyguanosine formation. The higher superoxide dismutase activity detected in mycorrhizal chicory roots could explain the benzo[a]pyrene tolerance of the colonized roots. Taken together, these results support an essential role of mycorrhizal fungi in protecting plants submitted to polycyclic aromatic hydrocarbon, notably by reducing polycyclic aromatic hydrocarbon-induced oxidative stress damage.     

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange

C57B1/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5 mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57B1/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction of by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.     

Mechanism of inactivation of sheep liver cytoplasmic aldehyde dehydrogenase by disulfiram.

Stoicheiometric amounts of [14C]disulfiram react rapidly with sheep liver cytoplasmic aldehyde dehydrogenase to give loss of catalytic activity and incorporation of the expected amount of radioactivity. In a subsequent slower reaction the label is lost from the enzyme without re-emergence of enzymic activity. The results imply that in vivo disulfiram may act as an oxidation-reduction catalyst for the inactivation of aldehyde dehydrogenase.     

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme.

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatography of aldehydes and acids formed in monoamine oxidase-catalyzed reactions

A rapid, sensitive, and specific method for the determination of monoamine oxidase (MAO) activities toward different substrates is described. The assay is based on high-performance liquid chromatographic (HPLC) separation and electrochemical detection of the aldehyde or acid products. The aldehyde metabolic intermediates were observed to be quite stable in 0.1 N perchloric acid containing antioxidant and EDTA, and therefore can be used to measure the MAO activity of washed mitochondrial membrane and partially purified or purified MAO. Incomplete conversion of aldehyde to acid was observed when the amine substrates were incubated with the crude enzyme preparations. These aldehydes can be converted to corresponding acids by addition of yeast aldehyde dehydrogenase and beta-NAD and the acid can also be measured by HPLC-electrochemical detection. A deuterium isotope effect in the oxidation of p-[alpha,alpha-2H2]tyramine and [alpha,alpha-2H2]serotonin has been demonstrated by this method.     

Subcellular localization of acetaldehyde dehydrogenase in human liver

The subcellular distribution of aldehyde dehydrogenase activity was determined in human liver biopsies by analytical sucrose density-gradient centrifugation. There was bimodal distribution of activity corresponding to mitochondrial and cytosolic localizations. At pH 9.6 cytosolic aldehyde dehydrogenase had a lower apparent Kappm for NAD (0.03 mmol l-1), than the mitochondrial enzyme (Kappm NAD = 1.1 mmol l-1). Also, the pH optimum for cytosolic aldehyde dehydrogenase activity (pH 7.5) was lower than that for the mitochondrial enzyme activity (pH 9.0), and the cytosolic enzyme activity was more sensitive to inhibition by disulfiram in vitro. Disulfiram (40 mumol l-1) caused a 70% reduction in cytosolic aldehyde dehydrogenase activity, but only a 30% reduction in mitochondrial enzyme activity after 10 min incubation. The liver cytosol may therefore be the major site of acetaldehyde oxidation in vivo in man.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.)

The spontaneous levels of micronuclei in erythrocytes were established in embryos of the black-headed gull of two natural populations. In total 216 blood samples from the same number of individuals were examined. A statistically significant decrease in the number of spontaneous micronucleated erythrocytes was found after 13 days of incubation. We found no statistically significant difference in the spontaneous frequencies of micronucleated erythrocytes in the embryos of the two colonies studied, although they differed in anthropogenic load. Results of analysis of variance indicated that egg incubation time was the only variable significantly (P=0.0001) affecting spontaneous frequency of micronucleated erythrocytes in the embryos of black-headed gulls. We also took 78 eggs of different developmental stages from both colonies and exposed them for a further 24h to a dose of benzo[a]pyrene (30 microg per egg). After exposure to benzo[a]pyrene, the frequency of micronucleated erythrocytes was not increased in the embryos incubated for a total period of 13 days. A statistically significant increase in the number of micronucleated erythrocytes was recorded in the benzo[a]pyrene-treated embryos incubated for a total period of 14 days. Decrease in numbers of spontaneous micronucleated erythrocytes after the 13 day of incubation and increased levels of benzo[a]pyrene-induced micronuclei after the 13 day of incubation were discussed to be caused by changes in spleen and liver function in advanced developmental stages of the embryo.     

Bacterial mutagenicity of two cyclopentafused isomers of benzpyrene

Two novel cyclopentafused polycyclic aromatic hydrocarbons, naphtho(1,2,3-mno)acephenanthrylene (cyclopenta benzo[e]pyrene) and naphtho(2,1,8-hij)acephenanthrylene (cyclopenta(ij)benzo[a]pyrene) were evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. Both compounds required S9 metabolic activation, and showed optimal activity at low S9 concentrations (below 0.6 mg/plate). Both compounds induced frameshift and base-pair substitution mutations, being active in strains TA98, TA100, TA1537, TA1538 and TA104, but not in strain TA1535. Cyclopenta(ij)benzo[a]pyrene was more active than cyclopentabenzo[e]pyrene, and both were more potent than their parent ring systems, benzo[a]pyrene and benzo[e]pyrene, respectively. Cyclopenta(ij)benzo[a]pyrene was more active in strain TA104 than in TA100 or TA98 (250-470, 340 and 80-100 rev/nmole) as was benzo[a]pyrene (120, 70 and 40 rev/nmole respectively); cyclopentabenzo[e]pyrene was more active in TA100 than TA104 or TA98 (70 versus 50 and 40 rev/nmole), and benzo[e]pyrene showed a similar pattern (4, 3.5 and 0.6 rev/nmole). The relative potencies of the four compounds are in accord with predictions based on perturbational molecular orbital calculations. The peak of activity at low S9 concentrations is consistent with epoxidation at the cyclopentafused ring being the major route of metabolic activation for both these cyclopentafused compounds.     

Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase

Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the enzyme with maximal velocities between 20-80 nmol/min per mg enzyme and Km values in the micromolar range. K-region dihydrodiols were not accepted as substrates. Dihydrodiols situated at the terminal ring of an anthracene-type structure such as benz[a]anthracene-8,9-dihydrodiol as well as the corresponding dihydrodiol epoxides were also not oxidized by DDH at measurable rates. The results provide evidence for a detoxifying role of DDH in the metabolism of the chemical carcinogens benz[a]anthracene, chrysene and dibenz[a, h]anthracene.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.     

Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.     

Mechanism of action of uridine diphoglucose dehydrogenase. Evidence for an essential lysine residue at the active site

The oxidation of UDP-glucose by the enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) from beef liver has been shown to proceed via the enzyme-bound intermediate, UDP-alpha-D-glyco-hexodialdose. The enzyme does not release this aldehyde, nor can it be trapped by reaction with hydroxylamine, thiosemicarbazide, or cyanide. Tight binding of the intermediate aldehyde can be explained by the recent observation that the essential thiol group of the enzyme forms a thiohemiacetal with the aldehyde during the course of the reaction. However, an enzyme preparation with the essential thiol derivatized with cyanide will still not release the aldehyde, indicating an additional as yet unknown binding mechanism. Derivatization ([14C]formaldehyde, followed by NaBH4 reduction) of 6 of the approximately 168 lysine residues per enzyme molecule (of six catalytic subunits) results in destruction of 47% of the enzyme activity, suggesting the involvement of an essential reactive lysine in the mechanism. Preincubation of the enzyme with UDP-glucose decreases both the loss of activity and incorporation of the label, indicating that this lysine is in the vicinity of the active site. Acid hydrolysis of the labeled preparation, followed by paper chromatography, shows that the label has a mobility, in the system used, that is identical with lysine. Elution of this spot followed by chromatography on Aminex A-5 resin showed that it contained the expected mixture of epsilon-N-methyl lysines. When enzyme that has its essential thiol derivatized with cyanide is incubated with UDP-[14C]glucose and NAD+, and then reduced with NaB3H4, a stable enzyme complex is formed which contains both labels. Acid hydrolysis of this preparation, followed by either two-dimensional paper chromatography or separation in an amino acid analyzer, results in both labels appearing in the position of lysine. It is evident that the enzyme oxidizes the UDP-[14C]glucose to the corresponding aldehyde which occurs as the Schiff's base with an essential lysine. This is then reduced by the NaB3H4 to form a secondary amine which is stable toward hydrolysis and migrates with lysine in separation procedures. As would be predicted, the enzyme can be similarly labeled by treatment with UDP-alpha-D-gluco-hexodisidose alone, followed by NaB3H4 reduction. The same hydrolysis product results from this procedure, and it behaves identically with the product formed by treating alpha-N-acetyl lysine with UDP-alpha-D-gluco-hexodialdose, reducing with NaBH4, and then hydrolyzing. This substance appears to be N5-((5-formyl-2-furanyl)methyl)lysine. When chromatographed on Aminex A-5, both the model compound and enzyme hydrolysate gave peaks corresponding to free lysine and the proposed derivative. Evidence is presented that the oxidation of UDP-glucose to the aldehyde is a concerted reaction involving the formation of the Schiff's base, rather than the formation of the aldehyde with the subsequent formation of the Schiff's base...     

Circular dichroism studies on the binding of type I substrates and reverse type I compounds to rabbit liver microsomal cytochrome P-450.

Liver microsomes from control, 3-methylcholanthrene-treated, and phenobarbital-treated New Zealand White rabbits were examined for differences detectable by circular dichroism (CD) spectroscopy. Addition of the Type I substrate cyclohexane to phenobarbital microsomes decreases the negative ellipticity at about 418 nm and concomitantly increases the negative ellipticity at about 395 nm. Cyclohexane added to microsomes from control or 3-methylcholanthrene-treated animals shows little or no CD changes in these wavelength regions. The effect by cyclohexane is completely reversed by the subsequent addition of butanol-1. Addition of benzo[a]pyrene to phenobarbital microsomes also decreases the negative ellipticity at about 418 nm, and this effect can be completely reversed with the subsequent addition of butanol-1. The ellipticity at about 395 nm is reversed in sign and is markedly increased by benzo[a]pyrene, however, and this effect is not changed with the subsequent addition of butanol-1. Restoring the cyclohexane- or benzo[a]pyrene-induced changes by the subsequent addition of alcohol is proportional to the aliphatic chain length, with 4 or more carbon atoms being maximally effective. Primary alcohols inhibit aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity of phenobarbital microsomes, and the inhibitory effect is enhanced with increasing chain length of the alcohols; 4 or more carbon atoms being maximally effective. Stimulation of monooxygenase metabolism of cyclohexane or benzo[a]pyrene by NADPH results in restoration of the negative ellipticity band at about 418 nm, whereas the ellipticity peak at about 395 nm remains unchanged. More negative ellipticity at about 210 and 222 nm is found in phenobarbital microsomes than in control or 3-methylcholanthrene microsomes and cyclohexane addition in vitro increases these negative ellipticity peaks in phenobarbital microsomes but not in control or 3-methylcholanthrene microsomes.These results show that with CD studies one can detect directly both high spin (negative ellipticity peak at 385–395 nm) and low spin (negative ellipticity peak at about 418 nm) P-450 iron in liver microsomes from control, 3-methylcholanthrene-treated, or phenobarbital-treated rabbits. These data are consistent with a weak ligand such as oxygen, rather than a stronger ligand such as nitrogen, in the sixth position of 6-coordinated (low spin) ferric iron in P-450 in vivo. Type I substrates such as cyclohexane or benzo[a]pyrene, when bound to P-450, change low spin P-450 iron to the high spin state. Cyclohexane-bound high spin P-450 iron in vitro is more easily converted to low spin iron by butanol-1 than is benzo[a]pyrene-bound high spin P-450 iron. Liver microsomal proteins from phenobarbital-treated rabbits have a higher helical content than those from either control or 3-methylcholanthrene-treated rabbits. Cyclohexane addition in vitro increases this helical character only in phenobarbital microsomes, indicating that one or more forms of phenobarbital-induced P-450 apoproteins is (are) more specific for cyclohexane binding and metabolism than control or 3-methylcholanthrene-induced forms of P-450.