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W F Bosron

Publications and source records attributed to W F Bosron.

90 records · Page 5Linked to original sources
PubMed1980

Rate-determining factors for ethanol metabolism in vivo during fasting.

Fasting produces a decrease in ethanol elimination rate of as much as 40% in rats and hamsters. In order to identify the biochemical process responsible for this change, the maximal activity of ADH, the cytosolic free NAD+/NADH ratio and the concentration of ethanol and acetaldehyde in liver were measured in both fed and 48 h fasted rats and hamsters after ethanol administration. While the maximal ADH activity of liver decreased 40% or more with fasting, only a small difference in NAD+/NADH ratio was observed between fed and fasted animals both 1 and 2 h following the administration of ethanol. Calculations of the steady-state rates of oxidation of ethanol by rat ADH revealed that the enzyme is rate-limited primarily by the concentration of free NAD+ in cytosol but that the steady-state rates of ethanol oxidation by ADH are 80-90% of the in vivo ethanol elimination rates. With fasting, the percentage decrease in steady-state rates was identical to that for ADH activity and for the in vivo elimination rate. These results indicate that changes in rates of oxidation of NADH and acetaldehyde contribute little toward the decrease in ethanol elimination rate associated with fasting but that the change in liver ADH activity or content is primarily responsible.

Acetaldehyde↗
PubMed1980

New molecular forms of human liver alcohol dehydrogenase: isolation and characterization of ADHIndianapolis.

The biochemical determinants of alcoholism and genetic correlates for the variability in man's response to alcohol have remained obscure until recently. The identification of genetically determined isoenzymes of alcohol dehydrogenase with different catalytic properties may bear importantly upon this problem. New molecular forms of human liver alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) have recently been identified in 16% of the liver specimens from an urban population from Indianapolis, Indiana [Bosron, W. F., Li, T.-K. & Vallee, B. L. (1979) Biochem. Biophys. Res. Commun. 91, 1549-1555]. The distinguishing features of these specimens were (i) they showed activity optima for ethanol oxidation at both pH 7.0 and 10.0 and (ii) they formed electrophoretic bands cathodic to the beta beta isoenzyme. From such livers, three new ADH forms have now been isolated, one of which has a single pH optimum at 7.0 and two of which have dual optima at pH 7.0 and 10.0. These new forms were designated ADHIndianapolis forms 1,2, and 3, respectively. They can be differentiated from previously described ADH isoenzymes, including the so-called "atypical" isoenzyme, by their electrophoretic mobility, pH optima, and Km for ethanol (approximately 60 mM at pH 7.5). Based upon the electrophoretic pattern of livers containing ADHIndianapolis and the mobility of the three isolated molecular forms, ADHIndianapolis may be the result of polymorphism at the ADH2 gene locus, which codes for the beta subunit.

Alcohol Oxidoreductases↗
PubMed1977

Effect of magnesium on the properties of zinc alkaline phosphatase.

Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1977

Isolation of pi-alcohol dehydrogenase of human liver: is it a determinant of alcoholism?

HUMAN LIVER ALCOHOL DEHYDROGENASE (ALCOHOL: NAD(+) oxidoreductase, EC 1.1.1.1), homogeneous by physicochemical criteria, has been available in quantity only recently [Lange, L. G. & Vallee, B. L. (1976) Biochemistry 15, 4681-4686]. Until now, the biochemical basis of human alcohol metabolism had to be extrapolated from the properties and behavior of enzymes from other species, primarily horses and yeast. The biological determinants of human alcoholism have remained obscure, although recent evidence indicates a genetic predisposition, requiring delineation. A functionally distinct form of human liver alcohol dehydrogenase (ADH), which we have designated II-ADH, is provocative since, thus far, it seems to be unique to human beings. It has a high K(m) for ethanol and is remarkably insensitive (apparent K(I), 500 muM) to pyrazole and its derivatives, which are usually potent ADH inhibitors (K(I), 1 muM), a property that is the basis for the isolation of II-ADH. The affinity resin 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole-Sepharose binds all other known forms of ADH but not II-ADH, thereby separating it selectively by affinity chromatography. In turn, this has led to the establishment of its identity with that enzyme form which was previously known as the anodic band and characterized by a high K(m) for ethanol (20 mM at pH 7.5). The remarkable insensitivity of II-ADH to pyrazole inhibition has also permitted quantitation of its role in hepatic ethanol oxidation. At 5 mM ethanol, a saturating concentration for virtually all other forms of ADH, II-ADH contributes less than 15% to total ethanol oxidation. However, at intoxicating concentrations, e.g., 60 mM, it can account for as much as 40% of the total ethanol oxidation rate of liver, indicating a seemingly unique role for this enzyme form in ethanol elimination. Thus far, we have found the amount of II-ADH varies from liver to liver of individuals and is considerably more labile than the other molecular forms, phenomena whose inter- or independence requires further study. The isolation of human II-ADH advances efforts to recognize and understand biochemical mechanisms that may be biological determinants of alcoholism and alcohol-related disease states, now generally approached and managed largely as psychosocial disorders.

Alcohol Oxidoreductases↗
PubMed1975

Zinc and magnesium content of alkaline phosphatase from Escherichia coli.

Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-aton of magnesium per molecular weight 80,000 (Plocke, D.J., Levinthal, D., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared.

Alkaline Phosphatase↗
PubMed1975

Role of magnesium in Escherichia coli alkaline phosphatase.

Alkaline phosphatase of E. coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom of magnesium and 4.0 +/- 0.2 g-atom of zinc per molecule of molecular weight 89,000. Magnesium, the role of which has been unappreciated, significantly affects the function and structure of alkaline phosphatase containing either 2 or 4 g-atom of zinc per mole. Magnesium does not activate the apoenzyme but increases the activity of the enzyme containing 2 g-atom of zinc 4.4-fold and that of the enzyme containing 4 g-atom 1.2-fold. The results obtained with enzyme in which cobalt is substituted for zinc are analogous. Moreover, the absorption and electron paramagnetic resonance spectra of cobalt phosphatases reveal the effects of magnesium on cobalt coordination geometry. Addition of magnesium changes the spectral characteristics of the apoenzyme reconstituted with 2 g-atom of cobalt from predominantly octahedral to 4- or 5-coordinate geometry. These two classes of cobalt binding sites have been associated with catalysis and structure stabilization, respectively. Therefore, magnesium controls the occupancy of the catalytic and structural binding sites and modulates the resultant enzymatic activity. Hydrogen-tritium exchange was employed to determine the effects of magnesium on the conformational stability of phosphatase. Magnesium stabilizes the dynamic structural properties, both of apophosphatase and of enzyme containing 2 g-atom of zinc, which is further stabilized by 2 more zinc atoms. The role of magnesium and other metal ions in regulatory processes, only now beginning to be explored fully, will likely emerge as an important avenue for achievement of regulatory effects in metalloenzymes.

Alkaline Phosphatase↗
PubMedDate not supplied

Genetic polymorphism of human liver alcohol and aldehyde dehydrogenases, and their relationship to alcohol metabolism and alcoholism.

It is now widely accepted that the various pharmacologic and addictive consequences of alcohol consumption are related to the tissue concentration of ethanol or its metabolic products. The oxidative metabolism of ethanol in liver is principally catalyzed by alcohol dehydrogenase and aldehyde dehydrogenase. Both of these enzymes exist in multiple molecular forms, and genetic models have been proposed to account for the multiplicity of isoenzymes. Alcohol dehydrogenase subunits are encoded at five different gene loci, and genetic polymorphism occurs at two alcohol dehydrogenase loci. Variant isoenzymes produced at the two polymorphic alcohol dehydrogenase loci account for the differences in enzyme electrophoretic patterns observed among individuals. Some of these variant isoenzymes exhibit widely different kinetic properties, and this may account for the 2- to 3-fold variation in alcohol elimination rate among individuals. Since the protein sequence of several of the alcohol dehydrogenase subunits has been determined and several of the alcohol dehydrogenase genes has been cloned, some of the structural changes which give rise to differences in catalytic and electrophoretic properties are now known. Genetic polymorphism also occurs at the aldehyde dehydrogenase gene locus which encodes the mitochondrial low Km for acetaldehyde aldehyde dehydrogenase isoenzyme. The variant isoenzyme exhibits little or no catalytic activity. Individuals with this "null" variant have higher than normal blood acetaldehyde levels and exhibit an alcohol-flush reaction which appears to be a deterrent to heavy drinking and alcoholism.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMedDate not supplied

Effect of fasting on the activity and turnover of rat liver alcohol dehydrogenase.

Alcohol dehydrogenase activity in rat liver decreased with fasting to about 60% of the fed level, but the specific activities of the enzyme purified from livers of fed and 12- or 48-hr fasted animals were similar, 3.2-3.4 U/mg protein. Therefore, the decrease in enzyme activity with fasting should have resulted from a decrease in the amount of enzyme protein. Accordingly, the turnover of alcohol dehydrogenase was examined in fed and fasted rats. The fractional rate of enzyme synthesis (ks) in fed rats was determined by radioisotopic methods to be 0.13 day-1 and it increased to 0.18 day-1 after a 12- or 48-hr fast. The absolute rate of synthesis (V) and the fractional rate of degradation (kd) were calculated from these ks values and the total enzyme content in livers from animals that were fasted for 8 to 72 hr. After 48-72 hr of fasting, V decreased 16% and kd increased about 20% with respect to the fed values. Together, these changes accounted for the lowered enzyme activity in the fasted state. The rapid decrease in enzyme activity with fasting, t1/2 congruent to 16 hr, was found to be due to a rapid increase in kd from 0.14-0.16 day-1 in fed animals to 0.61 day-1 during the first 8 hr after the initiation of fast. Thereafter, kd decreased steadily to reach 0.18 day-1 after 48-72 hr of fasting.

Alcohol Dehydrogenase↗
PubMedDate not supplied

Alcohol and aldehyde dehydrogenase isoenzymes in North American Indians.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzyme phenotypes were determined in autopsy liver samples from 50 North American Indians from New Mexico. Forty-six of the 50 livers had sufficient ADH activity to allow phenotyping at the ADH2 and ADH3 loci. All 46 livers possessed the "typical" ADH2 1-1 phenotype. The frequency of the ADH3(2) allele was 0.59 and is the highest thus far reported in any racial population. All 50 livers possessed the ALDH I isoenzyme which exhibits the greatest anodic mobility on starch gel electrophoresis at pH 7.6. The results show that ADH and ALDH phenotypes among American Indians living in New Mexico are very similar to those of Caucasian populations and quite different from those of Orientals.

Adolescent↗
PubMedDate not supplied

Kinetic characterization of two classes of dog liver alcohol dehydrogenase isoenzymes.

In order to relate the catalytic properties of alcohol dehydrogenase (ADH), the rate-limiting enzyme for alcohol metabolism, with the pharmacokinetics of ethanol elimination in vivo, the multiple molecular forms of dog liver ADH were purified and their steady state kinetics investigated. Two different classes of ADH forms were identified by starch gel electrophoresis: the class I isoenzymes migrate to the cathode and the class II forms migrate to the anode. Three different patterns of the cathodic class I isoenzymes were identified in different liver specimens. Three molecular forms were observed for patterns A and C, and five for B. The two classes of isoenzymes were separated by affinity chromatography and purified by column chromatography. The three predominant class I isoenzymes, A1, B2, and C1, in type A, B, and C livers, respectively, were isolated by high performance cation-exchange chromatography. The steady state kinetic constants of the A1, B2, and C1 isoenzymes are similar, but differ substantially from those of the class II enzyme. The class II enzyme is much less sensitive to pyrazole inhibition, Ki = 2 mM, than the class I forms, Ki = 0.6 microM. Methanol is not a substrate for the class II enzyme, whereas it is oxidized by the class I isoenzymes. The class I isoenzymes exhibit a lower Km and substrate inhibition Ki for ethanol, 0.4 and 160 mM, respectively, than values for the class II enzyme, 10 and 610 mM, respectively. The properties of class I and II dog liver ADH are similar to those of the respective isoenzymes purified from human and monkey liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗