Enzymatic and structural differences between usual and atypical human liver alcohol dehydrogenases.

A Yoshida,C.C Impraim,I.Y Huang

doi:10.1016/s0021-9258(18)43291-7

Abstract

Enzymatic and structural differences between usual and atypical human liver alcohol dehydrogenases.

Highlights

The usual homodimer PlPland the atypical homodimer f12P2isozymes were purified to homogeneity, The specific activity of the atypical enzyme was severaltimes higher at p H 10,and 80 times higher at pH 8.5, than that of the usual enzyme
In the usual &PI enzyme, as in the horse enzyme, the catalytic Zn is expected to link to the sensitive cysteine at position 47, histidine at position 67, and cysteine, presumably at position 174, making up the active site
Genetic polymorphism of human liver alcoholdehydrogenase has been known for some time(1-4).Most Caucasians have t"huesual" type cytosol alcohol dehydrogenase (2), while nearly 90% of Japanese and presumably otherMongoloids have the "atypical" type (5, 6)

Summary

MATERIALS AND METHODS

Thephenotype of alcohol dehydrogenase was determined by (a)total enzyme activity of the extract,( b )pH activity profile, and (c)starch gel electrophoresis, as previously described (6, 10, 11).Homozygous usual and homozygous atypical livers were used for enzyme preparation. Enzyme and Protein Assay-Alcohol dehydrogenase activity was assayed in 30 mM glycine/NaOH buffer at thespecified pH at 25 "C. For the assay of crude extract, the reaction mixture containing 0.1 mM pyrazole to inhibitalcohol dehydrogenase activity was used asa control. Peptide Mapping-The lyophilized enzyme was S-carboxymethylated or S-aminoethylated bypreviously described methods (15, 161, and the sample was digested by trypsin (Sigma type XI, diphenyl carbamyl chloride-treated). Atypical Human Alcohol Dehydrogenase hydrolyzed in the presence of thioglycolic acid as previously described (18). For determination of cysteine (or cystine) content, a sample of protein was oxidized with performic acid and subjected to aminoacid analysis (19). In this case. 5m0M Tris-phosphate, pH 9.0, was used instead of 50 mM phosphate,pH 7.5, since the atypical isozyme did not bindwell to the affinity column at pH 7.5

RESULTS

H W 81 s

Findings

DISCUSSION