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W F Bosron

Publications and source records attributed to W F Bosron.

At least 73 records · Page 4Linked to original sources
PubMed1987

Catalytic properties of human liver alcohol dehydrogenase isoenzymes.

Human liver alcohol dehydrogenase (ADH) exists in multiple molecular forms which arise from the association of eight different types of subunits, alpha, beta 1, beta 2, beta 3, gamma 1, gamma 2, pi, and chi, into active dimeric molecules. A genetic model accounts for this multiplicity as products of five gene loci, ADH1 through ADH5. Polymorphism occurs at two loci, ADH2 and ADH3, which encode the beta and gamma subunits. All of the known homodimeric and heterodimeric isoenzymes have been isolated and purified to homogeneity. Analysis of the steady-state kinetic properties and substrate and inhibitor specificities has shown substantial differences in the catalytic properties of the isoenzymes. For example, the Km values for NAD+ and ethanol vary as much as 1,000-fold among the isoenzymes. Some of the differences in catalytic properties can be related to specific amino acid substitutions in the ADH isoenzymes.

Alcohol Dehydrogenase↗
PubMed1987

Genetic control of liver alcohol dehydrogenase expression in inbred mice.

The expression of the mouse Adh-1 gene which encodes the ADH-AA isoenzyme of liver alcohol dehydrogenase is under genetic control. The content of alcohol dehydrogenase in the C57BL inbred strain of mice is about twice that in DBA mice. In order to examine the mechanism for control of liver alcohol dehydrogenase expression in mice, we have isolated liver RNA from high and low enzyme activity strains and quantified the content of ADH mRNA with the ADH-A cDNA that was recently cloned in our laboratory. The content of ADH-A mRNA in liver correlates well with enzyme activity in the mouse strains; hence, the expression of Adh-1 appears to be under transcriptional control. A restriction fragment polymorphism was found in mouse liver DNA which correlates with the content of ADH-AA in the high and low ADH activity strains.

Alcohol Dehydrogenase↗
PubMed1986

Role of the pituitary and neonatal androgenic imprinting in the hormonal regulation of liver alcohol dehydrogenase activity.

Liver alcohol dehydrogenase activity is increased by thyroidectomy, orchidectomy, or hypophysectomy. We investigated the mechanisms of these hormonal effects by examining the effects of testosterone, dexamethasone and thyroid hormone on liver alcohol dehydrogenase activity in hypophysectomized rats and in cultured hepatocytes, and the effect of administration of androgens to neonatal female rats. Testosterone did not lower alcohol dehydrogenase activity in hypophysectomized rats, whereas dexamethasone and thyroxine produced moderate decreases in activity. Triiodothyronine reduced alcohol dehydrogenase activity of cultured hepatocytes from male and hypothyroid female rats in a dose-dependent fashion, confirming that thyroid hormone had pituitary-independent effects on the enzyme activity. Dexamethasone was required for the expression of alcohol dehydrogenase activity in cultured cells, and it increased the enzyme activity when present at supraphysiologic concentrations. Treatment of neonatal female rats with testosterone reduced the activity of the enzyme in adulthood. The difference in alcohol dehydrogenase activity in adult male and female rats appears to be determined in part by neonatal imprinting by androgens and in part by an effect of testosterone that is either mediated by or dependent upon the pituitary. Thyroid hormone reduces alcohol dehydrogenase activity by a direct effect on the liver.

Adrenalectomy↗
PubMed1986

Selective carboxymethylation of cysteine-174 of the beta 2 beta 2 and beta 1 beta 1 human liver alcohol dehydrogenase isoenzymes by iodoacetate.

The beta 1 beta 1 and beta 2 beta 2 human liver alcohol dehydrogenase isoenzymes differ by only one residue at the coenzyme-binding site; Arg-47 in beta 1 is replaced by His in the beta 2 subunit. Since Arg-47 is thought to facilitate the carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase by binding halo acids in a Michaelis-Menten complex prior to inactivation, the specificity and kinetics of modification of the two human liver beta beta isoenzymes with iodoacetate were compared. Both of the beta beta isoenzymes were inactivated by treatment with iodo[14C]acetate, and one Cys per subunit was carboxymethylated. Cys-174, which is a ligand to the active-site zinc atom in horse liver alcohol dehydrogenase, was selectively carboxymethylated in each of the human beta beta isoenzymes; less than 15% of the iodo[14C]acetate incorporated into the enzyme appeared in Cys-46. Therefore, the three-dimensional structure of the basic amino acids in the anion-binding site of the human beta beta isoenzymes appears to be different from that of horse liver alcohol dehydrogenase. The kinetics of alkylation are consistent with the formation of a Michaelis-Menten complex before inactivation of the isoenzymes. The average Ki values for iodoacetate were 10 and 16 mM for beta 1 beta 1 and beta 2 beta 2, respectively, and maximal rate constants for inactivation were 0.22 and 0.17 min-1, respectively. From these data, it can be concluded that there is a relatively minor effect of the substitution of His for Arg at position 47 on the kinetics of inactivation.

Alcohol Dehydrogenase↗
PubMed1986

Genetic variability of enzymes of alcohol metabolism in human beings.

Alcohol is eliminated from the body almost entirely by hepatic metabolism, first to acetaldehyde, then to acetate, and finally to carbon dioxide and water. The time course of elimination is best described by Michaelis-Menten kinetics, and rates of elimination following standard doses of ethanol vary among subjects as much as three-fold. Studies comparing rates of elimination in identical and fraternal twins have shown that about half of the variability is attributable to genetic factors. The principal enzymes responsible for ethanol metabolism are alcohol dehydrogenase and aldehyde dehydrogenase. The reaction catalyzed by alcohol dehydrogenase is the rate-limiting step of the pathway. Human livers contain multiple isoenzymes of alcohol dehydrogenase, which are dimeric molecules arising from the association of two subunits encoded by five different structural genes. Genetic polymorphism at two of these gene loci has been described, and all known homo- and heterodimeric forms of the isoenzymes have now been isolated and characterized. Notably, some of them differ quite strikingly in reactivity toward ethanol. Thus a basis for the genetic variability in alcohol metabolic rate can be found in the kinetic properties of the alcohol dehydrogenase isoenzymes. The efficient oxidation of acetaldehyde by hepatic aldehyde dehydrogenase is essential for ethanol oxidation to continue over time, because the equilibrium of the alcohol dehydrogenase reaction favors the conversion of acetaldehyde to ethanol. Acetaldehyde is a very toxic substance the removal of which makes possible the consumption of large quantities of ethanol frequently imbibed by alcoholics. There are also multiple molecular forms of aldehyde dehydrogenase in liver, and the mitochondrial form is the one principally responsible for acetaldehyde oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1985

Cloning and sequencing of cDNA encoding the complete mouse liver alcohol dehydrogenase.

The main ethanol-active alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) in mouse liver (ADH-AA) is similar in catalytic and molecular properties to horse liver ADH-EE and to the human class I ADHs. We have isolated cDNA clones encoding the entire mouse liver enzyme plus flanking regions. A mixture of 16 different oligonucleotides, each 14 bases long, was used to screen a liver cDNA library made from a DBA/2J mouse. A strongly hybridizing clone was found and identified as an ADH-encoding cDNA by partial DNA sequencing. This clone was used as a probe to identify others. Two overlapping cDNA clones together contained the entire protein-encoding region plus 100 nucleotides of the 5' noncoding region and 133 nucleotides of the 3' noncoding region culminating in a short poly(dA) tail. The amino acid sequence of the mouse liver enzyme deduced from this cDNA closely resembles that of horse liver ADH-E: 316 of 374 residues are identical, and 29 of the differences are conservative substitutions. The 5' region of this cDNA is interesting: the AUG that initiates the ADH polypeptide is preceded by an AUG that would encode the first amino acid of a tripeptide. Presumably termination of this tripeptide is followed by reinitiation at the AUG immediately preceding the sequence of the mature ADH polypeptide.

Alcohol Dehydrogenase↗
PubMed1985

Purification and characterization of human liver beta 1 beta 1, beta 2 beta 2 and beta Ind beta Ind alcohol dehydrogenase isoenzymes.

Mammalian alcohol dehydrogenases (ADH) are dimeric molecules and electrophoretically distinct isoenzymes appear in human liver. They arise from the combination of different subunits produced at five separate gene loci and polymorphic subunits are produced at two of the loci. Three different homodimeric alloenzymes, beta 1 beta 1, beta 2 beta 2 and beta Ind beta Ind, that are assigned to the ADH2 gene have been purified to homogeneity, and the study of their kinetic properties has revealed striking differences among them. Whereas the pH-optimum for ethanol oxidation of beta 1 beta 1 is 10.0-10.5, that of beta 2 beta 2 is 8.5-8.8 and that of beta Ind beta Ind is 7.0-7.2. The Km of beta 1 beta 1 for ethanol at pH 7.5 is low, 49 microM, in contrast to values for beta 2 beta +2 and beta Ind beta Ind, 0.94 and 64 mM. Km and Ki values for NAD are 7.4 and 90 microM for beta 1 beta 1, 180 and 340 microM for beta 2 beta 2 and 490 and 1300 microM for beta Ind beta Ind. The activity of beta 1 beta 1 is stimulated by 40 mM NaCl but that of beta 2 beta 2 is not. Vmax values for ethanol oxidation range from 9.2 min-1 for beta 1 beta 1 to 400 and 560 min-1 for beta 2 beta 2 and beta Ind beta Ind. Both beta 1 beta 1 and beta 2 beta 2 are inactivated by iodoacetate with the formation of one S-carboxymethylcysteine per subunit; however, the second-order rate constant for inactivation of beta 1 beta 1 is four times that of beta 2 beta 2. These differences in the properties of beta 1 beta 1 and beta 2 beta 2 are consistent with the recently reported substitution of His in beta 2 for Arg 47 in beta 1 at the anion binding center of the active site. The active site sequence of the beta Ind subunit has not yet been determined.

Alcohol Oxidoreductases↗
PubMed1984

Human liver alcohol dehydrogenase: purification and kinetic characterization of the beta 2 beta 2, beta 2 beta 1, alpha beta 2, and beta 2 gamma 1 "Oriental" isoenzymes.

Four alcohol dehydrogenase isoenzymes with "atypical" pH optima for ethanol oxidation at 8.8 were isolated from Japanese livers with the homozygous ADH2 2-2 and the heterozygous ADH2 2-1 phenotypes. Agarose gel isoelectric focusing patterns after dissociation--recombination of three isoenzymes purified from the homozygous livers indicate that they are beta 2 beta 2, alpha beta 2, and beta 2 gamma 1. A fourth isoenzyme, purified from livers with the heterozygous phenotype by agarose-hexane--AMP affinity chromatography, was identified as beta 2 beta 1 by dissociation-recombination studies. The kinetic properties of the three heterodimers, beta 2 beta 1, alpha beta 2, and beta 2 gamma 1, are intermediate between those of the respective homodimers, suggesting that the two subunits act independently. Product inhibition studies indicate that beta 2 beta 2 obeys an ordered sequential mechanism, as do the alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2 homodimers which have the "typical" pH optimum for ethanol oxidation at pH 10.0-10.5. The kinetic constants of beta 2 beta 2 differ substantially from those of the other homodimers. At pH 7.5, the Vmax for ethanol oxidation of beta 2 beta 2 is 5-40 times higher than that of alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. The Km and Ki values of beta 2 beta 2 for NAD+ and NADH are also considerably higher than those of the other homodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1984

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity.

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The Km values for ethanol and NAD+, approximately 0.4 and 0.03 mM, respectively, of enzyme purified from both strains are similar. Moreover, the Ki for NADH, 1 microM, the pH optimum for ethanol oxidation, 10.5, and the Vmax for ethanol oxidation, 160 min-1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.

Alcohol Dehydrogenase↗
PubMed1984

Polymorphism of human liver alcohol dehydrogenase: identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing.

Liver homogenate-supernatants from most Japanese exhibit an "atypical" pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the "typical" pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with beta 2 subunits while typical livers contain isozymes with beta 1 subunits, both produced by the ADH2 gene. Because it is difficult to differentiate the atypical ADH2 2-2 phenotype from the ADH2 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The beta beta isozymes in A1 and A2 livers were purified. Type A1 livers contained a single beta beta isozyme with an atypical pH-rate profile; it was designated beta 2 beta 2. Three beta beta isozymes were isolated from A2 livers, two of which corresponded to beta 1 beta 1 and beta 2 beta 2. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated beta 2 beta 1. Type A1 livers are, therefore, the homozygous ADH2 2-2 phenotype, and type A2 livers, the heterozygous ADH2 2-1 phenotype. The ADH2 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH2 2-1 phenotype, in 31%. Accordingly, the frequency of ADH2(2) was 0.68.

Adult↗
PubMed1984

Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme.

Alcohol Dehydrogenase↗
PubMed1983

Steady-state kinetic properties of purified rat liver alcohol dehydrogenase: application to predicting alcohol elimination rates in vivo.

The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.

Alcohol Dehydrogenase↗
PubMed1983

Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase.

Ten, electrophoretically distinct, molecular forms of alcohol dehydrogenase have been isolated from a single human liver by affinity and ion-exchange chromatography. The starch gel electrophoresis patterns after the dissociation-recombination of the forms are consistent with the hypothesis that they arise from the random combination of alpha, beta 1, gamma 1, and gamma 2 subunits into six heterodimeric and four homodimeric isoenzymes. Large differences in kinetic properties are observed for the homodimeric isoenzymes, alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. At pH 7.5, the Km value of beta 1 beta 1 for ethanol is 0.049 mM and that of alpha alpha is 4.2 mM. Forms gamma 1 gamma 1 and gamma 2 gamma 2 do not obey Michaelis-Menten kinetics at pH 7.5 but exhibit negative cooperativity with Hill coefficients of 0.54 and 0.55 and [S]0.5 values of 1.0 and 0.63 mM, respectively. However, all isoenzymes display Michaelis-Menten kinetics for ethanol oxidation at pH 10.0 with Km values ranging from 1.5 to 3.2 mM. The maximum specific activity of beta 1 beta 1 is considerably lower than that of the other three homodimers at both pH 7.5 and 10.0. The Km values of the four homodimers for NAD+ at pH 7.5 range from 7.4 to 13 microM and those for NADH, from 6.4 to 33 microM. Ki values for NADH range from 0.19 to 1.6 microM. At pH 7.5, the kinetic properties of alpha alpha and beta 1 beta 1, prepared in vitro from dissociated and recombined alpha beta 1, are similar to those of the native homodimers. The forms gamma 1 gamma 1 and gamma 2 gamma 2, prepared from dissociated and recombined alpha gamma 1 and beta 1 gamma 2, respectively, exhibit negative cooperativity with Hill coefficients that are similar to those seen with the respective native homodimers.

Alcohol Dehydrogenase↗
PubMed1983

Human liver alcohol dehydrogenase: ADH Indianapolis results from genetic polymorphism at the ADH2 gene locus.

New molecular forms of human liver alcohol dehydrogenase (ADH), collectively designed ADH Indianapolis (ADHInd), were recently discovered in 29% of liver specimens from Black Americans [Bosron, W. F., Li, T.-K., and Vallee, B. L. (1981). Proc. Natl. Acad.Sci. USA 77:5784]. Three different ADHInd phenotypes have now been identified by starch gel electrophoresis, and four ADHInd enzyme forms isolated by affinity and ion-exchange chromatography. The most cathodic ADHInd form has a single pH optimum at 7.0 for ethanol oxidation and is a homodimer of a newly discovered subunit, as evidenced by dissociation--recombination studies. The remaining three purified ADHInd forms have dual pH optima for ethanol oxidation at 7.0 and 10.0 and generate two new bands on starch gel electrophoresis after dissociation-recombination. They appear to be heterodimers of this new subunit with the known subunits, alpha, beta 1, and gamma 1. Based on the occurrence of these four ADHInd isozymes and isozymes containing beta 1 subunits in the homogenate supernatants of 135 livers, we conclude that ADHInd results from polymorphism at the ADH2 locus, with the variant ADH2Ind allele coding for the beta Ind subunit. The frequency of ADH2Ind was 0.16 in Black Americans. The frequency of the ADH31 and ADH32 alleles also differed in these two populations.

Alcohol Dehydrogenase↗
PubMed1983

Relationship between kinetics of liver alcohol dehydrogenase and alcohol metabolism.

Since alcohol dehydrogenase (ADH) catalyzes the rate-limiting step for ethanol metabolism, knowledge of the steady-state kinetics of ADH in liver is fundamental to the understanding of the pharmacokinetics of ethanol elimination. Accordingly, we have determined the kinetic properties of purified ADH isoenzymes in rat and human liver. At low ethanol concentrations, rat liver ADH obeys the Theorell-Chance mechanism and the equation predicts that activity in vivo is limited below Vmax mainly by NADH inhibition. At ethanol concentrations above 10 mM, substrate inhibition, consistent with the formation a dead-end ADH-NADH-ethanol complex, also becomes a rate-limiting factor. ADH activity, calculated from this equation and the concentrations of substrates and products present in liver during ethanol oxidation, agrees well with ethanol elimination rates measured in vivo. With human liver ADH, large differences are observed in the kinetic properties of 5 homodimeric isoenzymes: gamma 1 gamma 1 and gamma 2 gamma 2 exhibit negative cooperativity for ethanol saturation, while alpha alpha, beta 1 beta 1 and beta ind beta ind obey Michaelis-Menten kinetics. At pH 7.5, Km values for ethanol and Vmax values range 0.048 mM and 9 min-1 for beta 1 beta 1 to 64 mM and 560 min-1 for beta ind beta ind, respectively. Therefore, individuals with different ADH phenotypes should display different ethanol elimination profiles.

Alcohol Dehydrogenase↗
PubMed1981

Simian liver alcohol dehydrogenase: isolation and characterization of isoenzymes from Saimiri sciureus.

The alcohol dehydrogenase (ADH) of squirrel monkey liver can be purified and separated into pyrazole-insensitive and pyrazole-sensitive isoenzymes by affinity chromatography. This is the first demonstration of two functionally distinct classes of ADH in a species other than man. The inhibition of the two enzyme fractions by 4-methylpyrazole is analogous to that observed for the corresponding isoenzymes of human liver. Similarly, the substrate specificity of the pyrazole-insensitive form is more limited and its Km for ethanol (4 mM at pH 7.5) and acetaldehyde (11 mM at pH 7.0) is larger than that of the pyrazole-sensitive isoenzymes. However, their physicochemical and compositional characteristics, i.e., molecular weight, zinc content, and dimeric structure, are all virtually identical with those of other mammalian alcohol dehydrogenases studied thus far. Zinc is essential for the enzymatic function of both molecular forms as demonstrated by inhibition with chelating agents.

Alcohol Oxidoreductases↗