PURIFICATION, IDENTIFICATION AND CHARACTERIZATION OF 'ATYPICAL' HUMAN LIVER ALCOHOL DEHYDROGENASE ISOENZYMES (JAPANESE).

摘要

Human livers contain multiple molecular forms of alcohol dehydrogenase; those in Class I arise from the random association into active dimers of three different subunit chains, (alpha),(beta), and (gamma), coded by three structural genes, ADH(,1), ADH(,2) and ADH(,3), respectively. Liver homogenate-supernatants from most Japanese are known to exhibit an "atypical" optimum for ethanol oxidation at pH 8.8 instead of 10.5 as is normally observed for mammalian alcohol dehydrogenases. It has been proposed that this "atypical" phenotype arises from a variant subunit (beta)(,2), with (beta)(,1) being the normal form. Because starch gel electrophoresis cannot differentiate homozygous and heterozygous "atypical" phenotypes, an agarose isoelectric focusing procedure was developed that clearly defined the ADH(,2) 2-2 and ADH(,2) 2-1 phenotypes in Japanese livers. The isoenzymes with the "atypical" pH-rate profile were purified. Three of these were isolated from type ADH(,2) 2-2 livers and dissociation-recombination experiments demonstrated that they are (beta)(,2)(beta)(,2), (alpha)(beta)(,2) and (beta)(,2)(gamma)(,1). A fourth isoenzyme with "atypical" pH-rate profile, (beta)(,2)(beta)(,1), was purified from type ADH(,2) 2-1 livers. The ADH(,2) 2-2 phenotype was found in 53 percent of 194 Japanese livers and the ADH(,2) 2-1 phenotype, in 31 percent. Accordingly, the frequency of the ADH(,2)('2) allele was 0.68.;The specific enzymatic activities of (beta)(,2)(beta)(,1), (alpha)(beta)(,2) and (beta)(,2)(gamma)(,1) were found to be intermediate(, )between those of the respective homodimers, and the kinetics of (beta)(,2)(beta)(,1) for NADH oxidation by acetaldehyde were biphasic; therefore, the two subunits appear to act independently. Product(, )inhibition studies indicated that (beta)(,2)(beta)(,2) obeys an ordered bibi mechanism. At pH 7.5, the V(,max) of (beta)(,2)(beta)(,2) for ethanol oxidation and its K(,m)'s and K(,i)'s for NAD('+) and NADH were 3-51 times larger(, )than those of (beta)(,1)(beta)(,1), (alpha)(alpha), (gamma)(,1)(gamma)(,1) and (gamma)(,2)(gamma)(,2). The (beta)(,2)(beta)(,2) and (beta)(,1)(beta)(,1) were stoichiometrically modified by iodoacetate forming S-carboxymethylcysteine at the active site and resulting in inactivation.(, )The second order rate constant of inactivation for (beta)(,1)(beta)(,1) was 4-times larger than that for (beta)(,2)(beta)(,2). The above results are consistent with the recently proposed substitution of histidine in the (beta)(,2) subunit for arginine in (beta)(,1) at the coenzyme anion binding domain of the active site.