Substrate For Alcohol Dehydrogenase Research Articles

Hen serum albumin hydrolyses an organophosphorus compound

Biotransformation of 2-methoxyethanol (2-ME) by alcohol and aldehyde dehydrogenases is an established factor in the toxicity of this useful solvent. Little is known about potential capacity for 2-ME biotransformation by testis or other target tissues. We detected appreciable capacity for 2-ME biotransformation by alcohol dehydrogenase in testes from Sprague-Dawley rats. However, kinetic analysis showed a 6-fold lower affinity for 2-ME by alcohol dehydrogenase of testis compared to liver. 2-ME biotransformation was also detected in testes from Wistar rats and one strain of mice but not in testes from hamsters, guinea pigs, rabbits, dogs, cats or humans. Testes from all these species readily converted the aldehyde metabolite of 2-ME to 2-methoxyacetic acid. Hepatic capacities for 2-ME biotransformation by alcohol dehydrogenase varied from 22 to 2.5 μmol/mg prot/min with a species rank order of: hamsters ⪢ rats = mice>guinea pigs = rabbits. There was no consistent concordance between activities for 2-ME versus ethanol, the prototype substrate for alcohol dehydrogenase, which could reflect substrate preferences of different isozymes. Species differences between rats and hamsters were also found for testicular and hepatic biotransformation of the glycol ethers, 2-ethoxyethanol and 2-butoxyethanol. Although species differences in capacity for 2-ME biotransformation were found, the observations do not provide an explanation for reported species and strain differences in susceptibility to 2-ME toxicity.

Ethanol inhibition of retinoic acid synthesis as a potential mechanism for fetal alcohol syndrome

Retinoic acid (RA) is known to act as a signaling molecule during embryonic development, but little is known about the regulation of RA synthesis from retinol. The rate-limiting step in RA synthesis is the oxidation of retinol, a reaction that can be catalyzed by alcohol dehydrogenase (ADH). Ethanol is also a substrate for ADH, and high levels of ethanol inhibit ADH-catalyzed retinol oxidation. This has prompted us to hypothesize that ethanol-induced defects observed in fetal alcohol syndrome involve ethanol inhibition of ADH-catalyzed RA synthesis. Here, we have examined the effect of ethanol on RA levels in cultured mouse embryos by using a bioassay. Treatment with 100 mM ethanol, but no 10 mM, led to a significant decrease in RA detection in 7.5-day-old embryos. Using whole-mount in situ hybridization, we detected mRNA for class IV ADH, but not ethanol-active cytochrome P450 2E1, in 7.5- and 8.5-day-old embryos, indicating that an ADH-linked pathway exists at these stages for metabolizing retinol and ethanol. Thus, the observed ethanol-induced reduction in RA may be caused by ethanol inhibition of retinol oxidation catalyzed by class IV ADH. In our postulated mechanism for fetal alcohol syndrome, this enzyme may well play a crucial role.

Species differences in testicular and hepatic biotransformation of 2-methoxyethanol

Biotransformation of 2-methoxyethanol (2-ME) by alcohol and aldehyde dehydrogenases is an established factor in the toxicity of this useful solvent. Little is known about potential capacity for 2-ME biotransformation by testis or other target tissues. We detected appreciable capacity for 2-ME biotransformation by alcohol dehydrogenase in testes from Sprague-Dawley rats. However, kinetic analysis showed a 6-fold lower affinity for 2-ME by alcohol dehydrogenase of testis compared to liver. 2-ME biotransformation was also detected in testes from Wistar rats and one strain of mice but not in testes from hamsters, guinea pigs, rabbits, dogs, cats or humans. Testes from all these species readily converted the aldehyde metabolite of 2-ME to 2-methoxyacetic acid. Hepatic capacities for 2-ME biotransformation by alcohol dehydrogenase varied from 22 to 2.5 μmol/mg prot/min with a species rank order of: hamsters ⪢ rats = mice>guinea pigs = rabbits. There was no consistent concordance between activities for 2-ME versus ethanol, the prototype substrate for alcohol dehydrogenase, which could reflect substrate preferences of different isozymes. Species differences between rats and hamsters were also found for testicular and hepatic biotransformation of the glycol ethers, 2-ethoxyethanol and 2-butoxyethanol. Although species differences in capacity for 2-ME biotransformation were found, the observations do not provide an explanation for reported species and strain differences in susceptibility to 2-ME toxicity.

Effect of flood stress on morphology and anaerobic metabolism of Momordica charantia

Flooding induced several morphological and physiological changes in bitter melon ( Momordica charantia L.), a flood-intolerant plant; hypocotyl cross-sectional area and numbers of adventitious roots increased significantly after 4 days of flooding. On the first day of flooding, aerobic respiratory rate of flooded roots dropped to 42% of the control level. Activities of alcohol dehydrogenase (ADH) and endogenous concentrations of ethanol increased gradually, reaching a maximum level on the third day of flooding. Concomitantly, an isozymic form of ADH was shown to be induced. An increase in the apparent K m for the ADH substrate, acetaldehyde, was found to be positively correlated with the decline of endogenous concentrations of ethanol after the 3 day flood stress. A prolonged accumulation of ethanol in flood-stressed roots was shown to lead to death of the seminal root system.

Mutations of human class III alcohol dehydrogenase.

The residues lining the active site pocket of class III alcohol dehydrogenase differ markedly from those of class I and other classes. Two charged residues in the vicinity of the catalytic site are characteristic of class III. One is Arg115, which is at the outer part of the substrate-binding cleft and is associated with major enzymatic characteristics of class III, i.e. fatty acid activation (Moulis et al., 1991; Holmquist et al., 1993) and glutathione-de-pendent formaldehyde dehydrogenase activity (Engeland et al., 1993). The other charged residue is Asp57, located in the middle part of the substrate pocket. In an attempt to study the relative importance of residues involved in differentiation of classes, we have by site-directed mutagenesis examined not only the function of Asp57, but also the roles of Tyr93 and Thr48 of class III (Estonius et al., 1994). The charge and polarity at positions 57 and 93 were altered by Asp57Leu and Tyr93Phe substitutions, respectively. As opposed to Asp in class III, Leu is found at position 57 in most class I alcohol dehydrogenases, and in contrast to Tyr in class III, Phe or Ala are found at position 93. Therefore, a combination of the Asp57Leu and Tyr93Phe exchanges in one mutant is of interest in order to see if a class I alcohol dehydrogenase substrate pocket, and class I enzymatic properties, can be mimicked by altering the active site structure in a direction toward those of class I enzymes. In addition, to check for differences in the oxidation of short-chain aliphatic alcohols contra S-hy-droxymethylglutathione (HMGSH), we have replaced Thr48 with Ala, a mutation deleterious to class I alcohol dehydrogenase activity (Hoog et al., 1992).

Redox Activity and Alcohol-mediated Energization of the Plasma Membrane of Plant Cells

Summary Medium acidification accompanying ferricyanide-induced plasma membrane redox activity of leaves of the fresh-water plant Egeria densa was shown to be indirectly and not stoichiometrically coupled with the ferricyanide reduction rate. The ATPase inhibitors DCCD and erythrosine B had no effect on the ferricyanide reduction rate whereas they essentially inhibited medium acidification. Ethanol (0.1-1% = 17-170 mol m -3 ) caused the energization of the plasma membrane in darkadapted Egeria leaves. This became evident by the strong ethanol-induced hyperpolarization of the plasma membrane and the stimulated net proton efflux out of leaves measured as medium acidification. Also, the ferricyanide-reducing activity (FRA) as well as the uptake of a aminoisobutyric acid (Affi) and sucrose by Egeria leaves were significantly enhanced in the presence of ethanol. Ethanol-induced energization was, at least partly, inhibited by the alcohol dehydrogenase inhibitor, pyrazol. Furthermore, alcohols that are substrates of alcohol dehydrogenase (1-propanol, 1-butanol) exhibit similar effects on membrane energization as ethanol, whereas alcohols that are hardly metabolized (methanol, 2-propanol) do only to a lesser extent. In epidermal strips of the monocot, Commelina communis , ethanol (1 %) significantly stimulated stomatal opening whereas no effect of the same concentration of methanol could be found. Using the example of fusicoccin, the significance of these results for the use of alcohols as solvents for phytoeffectors is discussed.

A Physiologically Based Pharmacokinetic Model for Nasal Uptake and Metabolism of Nonreactive Vapors

A PB-PK model has been developed for nasal nonreactive vapor uptake in the F344 rat which incorporates nasal enzyme distribution as well as nasal airflow patterns. Nasal tissue is separated into respiratory and olfactory mucosal areas with each area containing mucus, epithelial, and submucosal compartments. Metabolic activities are distributed among the compartments in accordance with published histochemical data, and intercompartmental transfer rate constants are based on molecular diffusivity. Two airflow paths are assumed: a lateral/ventral path which passes over only respiratory mucosa and a dorsal medial path which passes over both respiratory and olfactory mucosa. This model was fit to F344 rat nasal uptake data obtained for five vapors: acetone (an unmetabolized vapor), isoamyl alcohol (alcohol dehydrogenase substrate), ethyl acetate (carboxylesterase substrate), and o-xylene and bromobenzene (mixed function oxidase substrates). These vapors cover a wide span of uptake values ranging from a fractional uptake of less than 0.01 for xylene to 0.80 for isoamyl alcohol. The model accurately predicted the fractional uptake of these vapors, the maximal deviation between actual and predicted values being 0.04. The best fit for the data was obtained assuming: (1) an asymmetric blood flow pattern (with the majority of the blood flow being apportioned to the respiratory mucosa), and (2) only a small fraction of the total airflow (8%) following the dorsal medial pathway and penetrating to the olfactory mucosa. Since only a small fraction of the inspired air passes over the olfactory mucosa, a site with high metabolic activity, an inspiratory flow rate dependence on inspired vapor metabolism results. At low flows, olfactory metabolism is limited by low vapor delivery to that site, an effect that does not occur at high flows. This highlights the potential importance of regional nasal airflow and enzymatic activity patterns in influencing uptake and metabolism of inspired vapors and indicates the need for physiologically relevant models incorporating these factors.

A Spectrophotometric Assay for α-Ketoaldehydes Using Horse Liver Alcohol Dehydrogenase

α-Ketoaldehydes have been extensively employed as reagents for the chemical modification of arginine residues in proteins, and to probe for putative anion recognition sites. A major disadvantage in their use is instability, as α-ketoaldehydes are prone to hydration, oxidation, and polymerization. These reagents are typically supplied as the aldehyde hydrates which are relatively stable, but must be purified and standardized prior to analytical use. The known chemical, spectroscopic, and enzymatic methods for quantitating α-ketoaldehydes are not practical for routine analysis due principally to their high detection limits and sensitivity to interfering substances. Surprisingly, α-ketoaldehydes have not been reported as substrates for the alcohol dehydrogenases. We have discovered that phenylglyoxal and several related α-ketoaldehydes are good substrates for horse liver alcohol dehydrogenase (HLADH). The second order rate constants (kcat/Km) are within a factor of 10 of that for the reduction of acetaldehyde, a known good substrate for HLADH. The enzymatic reduction reaction is stoichiometric with the oxidation of NADH, resulting in a rapid, convenient, and sensitive method for the spectrophotometric quantitation of α-ketoaldehydes, with a submicromolar detection limit for phenylglyoxal. The sole product of phenylglyoxal reduction has been identified as α-hydroxyacetophenone. The ketone functional group is not reduced, and the enzymatic reaction is essentially irreversible as α-hydroxyacetophenone is not oxidized to phenylglyoxal by HLADH in the presence of NAD+.

Treatment of Ethylene Glycol Poisoning

In their recent article, Curtin et al 1 probably relied on a computer search to compile their bibliography. While computers and bibliographic software programs speed up literature searching, they do have their own pitfalls; chiefly, that the world's literature begins when the databases were started. Therefore, articles published earlier, are overlooked. I should like to point out that the use of ethyl alcohol to treat ethylene glycol poisoning goes back to 1963 when studies by Peterson et al 2 showed that ethylene glycol is a substrate of alcohol dehydrogenase and that ethyl alcohol inhibits its oxidation, thus preventing the conversion of ethylene glycol to toxic intermediates and oxalic acid. Moreover, in the same article, experiments carried out using squirrel monkeys proved that treatment of these animals with ethyl alcohol after supertoxic doses of ethylene glycol was successful. These studies were the basis for the first effective treatment of humans 3 that

Upper Respiratory Tract Metabolism of Inspired Alcohol Dehydrogenase and Mixed Function Oxidase Substrate Vapors Under Defined Airflow Conditions

AbstractNumerous xenobiotic-metabolizing enzymes are present in nasal tissues but little is known about the extent to which inspired vapors are metabolized in that site. In the current study, the metabolism of inspired isoamyl alcohol (an alcohol dehydrogenase substrate), and o-xylene, bromobenzene, and ferrocene (mixed function oxidase [MFO] substrates) was examined by quantitating the effects of metabolic inhibitor pretreatment on uptake of these vapors in the surgically isolated upper respiratory tract (URT) of the F344 rat and Syrian hamster under defined airflow conditions. Constant velocity unidirectional inspiratory flow rates of 35, 70, or 200 ml/min, and 50, 100, and 300 ml/min were used in the hamster and rat, respectively. In both species, URT deposition efficiency of isoamyl alcohol vapor was reduced by 3–6% by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole (p <.01), providing strong evidence that this vapor was metabolized by nasal alcohol dehydrogenase. Pretreatment w...

The catalytic specificity of liver alcohol dehydrogenase: vitamin A alcohol and vitamin A aldehyde activities.

Liver alcohol dehydrogenase (LADH, E.C.I. 1.1.1) is an NAD+/NADH dependent enzyme with a broad substrate specificity. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones, as well as the oxidation of certain aldehydes to their carboxylic acids (Jornvall, 1970; Branden, et al., 1975; Klinman, 1981; Eklund and Branden, 1983; Zeppezauer, 1986; Pocker, et al., 1986; Pettersson, 1987; Pocker, 1989). The enzyme’s primary and tertiary structures have been determined, as have its catalytic mechanisms, isozyme differences and evolutionary divergences (Eklund and Branden, 1987; Eklund, et al., 1987; Pocker, 1989, Light, et al., 1992). Although the physiological substrates of alcohol dehydrogenase are unknown, this enzyme catalyzes the interconversion of all-trans retinol, Vitamin A alcohol and all-trans retinal, Vitamin A aldehyde, Figure 1 (Pocker et al., 1987). In addition, liver alcohol dehydrogenase catalyzes the oxidation of all-trans retinal to the corresponding retinoic acid (Pocker et al., 1987).

Stimulation of germination of dormant oat (Avena sativa L.) seeds by ethanol and other alcohols

AbstractDormant dehusked Avena sativa L. (cv. Moyencourt) caryopses germinated easily at 10°C, but their germination was poor at 25 and 30°C. Ethanol overcame their dormancy and allowed germination at the last two temperatures when given continuously at concentrations ranging from 50 to 200 mm, or at higher concentrations at the beginning of imbibition. Ethanol also promoted germination in hypoxia. Other alcohols which were good substrates for alcohol dehydrogenase (ADH: EC 1.1.1.1), such as butanol-1, propanol-1 and 2-propen-1-ol, had the same stimulatory effect as ethanol, whereas alcohols which could not be oxidized by ADH (propanol-2 and methanol) did not improve germination. Salicylhydroxamic acid (SHAM) did not alter the stimulation of germination induced by ethanol, but 4-methylpyrazole (4-MP, an inhibitor of ADH) completely abolished this stimulation. The improvement of germination by alcohols which were good substrates for ADH was always associated with an increase in oxygen uptake by caryopses, whereas alcohols which were not ADH substrates did not enhance respiration. The stimulation of oxygen uptake induced by ethanol resulted in a decrease in CO2/O2 ratio and was suppressed by 4-MP, but ATP level in embryos was not modified until after the germination of caryopses. All the results obtained seem to demonstrate that the stimulatory action of ethanol on germination of dormant oat caryopses requires its metabolism through ADH and involves an activation of glycolysis and the Krebs cycle.

Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592

Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.

Generation of Reactive Oxygen Species and Reduction of Ferric Chelates by Microsomes in the Presence of a Reconstituted System Containing Ethanol, NAD+ and Alcohol Dehydrogenase

Many of the toxic metabolic actions of ethanol on the liver have been ascribed to the enhanced cellular production of NADH, which arises as a consequence of the oxidation of ethanol by alcohol dehydrogenase (ADH). Experiments were conducted to evaluate whether NADH generated from a reconstituted system containing ethanol plus NAD+ plus ADH could interact with ferric chelates to promote microsomal lipid peroxidation and generation of a hydroxyl radical (OH)-like species. In the presence of the reconstituted system and iron, microsomes produced.OH as assessed by the oxidation of .OH scavenging agents. This oxidation was inhibited by catalase and competitive.OH scavengers but not by superoxide dismutase. The ADH-dependent microsomal production of.OH was effectively catalyzed by ferric-EDTA and -diethylenetriamine pentaacetic acid (-DTPA), but not by ferric-ATP or -citrate. However, all these ferric chelates were reduced by the microsomes in the presence of the reconstituted system. Hydrogen peroxide (H2O2) was produced in the presence of ADH and appeared to be a limiting factor for the production of.OH. The reconstituted system also catalyzed microsomal lipid peroxidation, and the pattern of effectiveness of ferric chelates was opposite that of catalysis of.OH production. There was little effect by catalase, superoxide dismutase or dimethyl sulfoxide (DMSO) on the ADH-dependent microsomal lipid peroxidation. The reconstituted system was characterized with respect to dependence on NAD+ and ADH; ethanol could be replaced by other alcohols, which are substrates for ADH. Pyrazole, a potent inhibitor of ADH, blocked the ability of the reconstituted system to interact with iron and microsomes to produce reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorometric assays for isozymes of human alcohol dehydrogenase

Two new fluorogenic substrates for human alcohol dehydrogenase (ADH), 4-methoxy-1-naphthaldehyde (IA) and 6-methoxy-2-napthaldehyde (IIA), are described. The 2-naphthaldehyde derivative fluoresces in aqueous media with a quantum yield of 0.22 with an emission maximum at 450 nm, but the 1-naphthaldehyde shows only weak fluorescence. The corresponding alcohol reduction products, 4-methoxy-1-naphthalenemethanol (IB) and 6-methoxy-2-naphthalenemethanol (IIB), exhibit fluorescence in the near uv region with quantum yields of 0.36 and 0.26, respectively. The K m values for the individual homogenous class I ADH isozymes, with the above naphthaldehydes as substrates, range from 0.35 to 11.5 μ m. The k cat values range from 70 to 610 min −1 and are thus comparable to those for the best ADH substrates. Except for the β 1 β 1 isozyme, IA is the preferred substrate for class I ADH isozymes while IIA is rapidly reduced by class II (π-ADH). The sensitivity and specificity of the enzymatic assay with IA as substrate are demonstrated and provide the basis for the determination of class I ADH activity in human serum.

New perspectives in catalase-dependent ethanol metabolism.

The notion that catalase is a minor pathway of ethanol oxidation must be reexamined in view of recent work. Studies with aminotriazole demonstrate clearly that catalase can be the predominant pathway of ethanol metabolism. In addition, these studies illustrate that caution must be used in interpretation of work with aminotriazole unless the extent of inhibition of catalase is controlled carefully. Studies of rates of oxidation of butanol, a specific substrate for alcohol dehydrogenase, and methanol, a substrate for catalase, indicate that peroxidation via catalase supported by H2O2 formed by the peroxisomal beta-oxidation of fatty acids is the predominant pathway of alcohol oxidation in the fasted state.

Metabolic activation and detoxification of aliphatic alcohols in Drosophila melanogaster and Ceratitis capitata in relation to their alcohol dehydrogenase activity

Three Drosophila melanogaster mutant strains with normal alcohol dehydrogenase (ADH) levels (ADH +) and three strains with no ADH activity (ADH −) are used to evaluate the importance of ADH in the metabolic activation and detoxification of aliphatic alcohols and particularly methanol, ethanol, 1-pentyn-3-ol, and 2-cyclohexen-1-ol. Methanol toxicity is not influenced by ADH based on observations that methanol and deuteromethanol (CD 3OH) are not substrates for ADH and they are of similar toxicity to ADH + and ADH − strains. Ethanol, 1-pentyn-3-ol (and the related 3-penten-2-ol), and 2-cyclohexen-1-ol are oxidized >20-fold faster by homogenates of ADH + than of ADH − flies, in each case requiring NAD and converting the latter three secondary alcohols to the corresponding ketones identified by gas chromatography-mass spectrometry. Ethanol oxidation is a detoxification process based on two findings: ADH + flies are ∼10-fold less sensitive than ADH − flies to the lethal effects of ethanol, confirming earlier findings; introduction of deuterium into the methylene position (CH 3CD 2OH and CD 3CD 2OH versus CD 3CH 2OH and CH 3CH 2OH) decreases the rate of ADH oxidation by 3-fold leading to a 1.5-fold higher toxicity of the ethanol, a new observation. 1-Pentyn-3-ol oxidation is an activation reaction since this pentynol is 15-fold more toxic to ADH + than to ADH − flies. Surprisingly, the relatively high toxicity of 2-cyclohexen-1-ol is not dependent on ADH activity, with equal potency on ADH + and ADH − strains, or on 2-cyclohexen-1-one formation. The toxicity of these alcohols in adult Ceratitis capitata is generally similar to that in ADH + D. melanogaster and a 2- to 4-fold sex difference in Ceratitis ADH activity does not confer a significant sex difference in sensitivity to alcohol intoxication.

Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analogue of oxidized nicotinamide adenine dinucleotide.

The dinucleotide carbanicotinamide adenine dinucleotide (carba-NAD), in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide ribonucleoside moiety of NAD, has been synthesized and characterized enzymologically. The synthesis begins with the known 1-aminoribose analogue (+/-)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. The pyridinium ring is first introduced and the resultant nucleoside analogue specifically 5'-phosphorylated. Coupling the racemic carbanicotinamide 5'-mononucleotide with adenosine 5'-monophosphate produces two diastereomeric carba-NAD analogues which are chromatographically separable. Only one diastereomer is a substrate for alcohol dehydrogenase and on this basis is assigned a configuration analogous to D-ribose. The reduced dinucleotide carba-NADH was characterized by fluorescence spectroscopy and found to adopt a "stacked" conformation similar to that of NADH. The analogue is reduced by both yeast and horse liver alcohol dehydrogenase with Km and Vmax values for the analogue close to those observed for NAD. Carba-NAD is resistant to cleavage by NAD glycohydrolase, and the analogue has been demonstrated to noncovalently inhibit the soluble NAD glycohydrolase from Bungarus fasciatus venom at low concentrations (less than or equal to 100 microM).

Substrate specificity of human class I alcohol dehydrogenase homo- and heterodimers containing the beta 2 (Oriental) subunit.

In order to gain a better understanding of the metabolism of ethanol in Orientals, the kinetic properties of human alcohol dehydrogenase (ADH) isozymes containing the beta 2 (Oriental) subunit, i.e., alpha beta 2, beta 2 gamma 1, beta 2 beta 2, beta 2 gamma 2, as well as gamma 1 gamma 1, were examined by using primary and secondary alcohol substrates of various chain lengths and compared with those of the corresponding beta 1 (Caucasian) subunit containing isozymes already on record [Wagner, F. W., Burger, A. R., & Vallee, B. L. (1983) Biochemistry 22, 1857-1863]. With primary alcohols, these isozymes follow typical Michaelis-Menten kinetics with a preference for long-chain alcohols, as indicated by Km and kcat/Km values. The kcat values obtained with primary alcohols, except methanol, do not vary greatly, i.e., less than 3-fold, whereas the corresponding Km values span a 3600-fold range, i.e., from 26 microM to 94 mM, indicating that the specificity of these isozymes manifests principally in substrate binding. As a consequence, ethanol--which might be thought to be the principal in vivo substrate for ADH--is oxidized rather poorly, i.e., from 50- to 90-fold less effectively than octanol. Secondary alcohol oxidation by the homodimers beta 2 beta 2 and gamma 1 gamma 1 also follows normal Michaelis-Menten kinetics. Again, values of Km and kcat/Km reveal that both isozymes prefer long carbon chains. For all secondary alcohols studied, the Km and kcat values for beta 2 beta 2 are much higher than those for gamma 1 gamma 1, i.e., 25- to 360-fold and 6- to 16-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclohexanol and methylcyclohexanols: A family of inhibitors of hepatic HMGCoA reductase in vivo

Oral dosing of rats with cyclohexanol and methylcyclohexanols resulted in the inhibition of hepatic HMGCoA reductase. Neither cyclohexane or cyclohexane diols exerted any effects. Inhibition was not due to alcohol dehydrogenase mediated changes in redox state since 3,3′,5-trimethylcyclohexanol (TMC), a non substrate for alcohol dehydrogenase, was a potent inhibitor of HMGCoA reductase. Following a single dose of TMC there was no alteration in total hepatic HMGCoA reductase activity for more than 6 hr after which the enzyme activity was depressed in a dose-dependent manner. The normal diurnal rhythm of HMGCoA reductase was reduced in amplitude following TMC administration but the phase was unaltered and the t 1 2 for activity decay following the peak of activity was unaffected. Prior to the inhibitory effect of a TMC dose becoming apparent in total HMGCoA reductase activity we found that the expressed activity of the enzyme (after isolation in F − medium to suppress endogenous protein phosphatase) was depressed by 43%. The inhibitory effect of TMC on total HMGCoA reductase activity seen 8 hr or more after dosing was reflected by inhibition of sterol synthesis in liver measured in vivo after [ 3H]-H 2O administration.