CHARACTERIZATION OF S-HEXYLGLUTATHIONE-BINDING PROTEINS OF HUMAN HEPATOCELLULAR-CARCINOMA - SEPARATION OF ENOYL-COA ISOMERASE FROM AN ALPHA-CLASS GLUTATHIONE TRANSFERASE FORM

Recent studies have revealed binding of mitochondrial enoyl-CoA isomer ase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has b een suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylg lutathione-binding proteins of human hepatocellular carcinomas were ch aracterized to examine their identity. Supernatant fractions of carcin oma and surrounding tissues were applied to an affinity column, and bo und fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by tw o-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were elu ted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separ ated from each other. The 29 kDa protein exhibited a low but significa nt activity towards 1-chloro-2,4-dinitrobenzene (4.25 mu mol/min per m g of protein) and reacted with anti-(GST A1-2) antibody, suggesting th at it is a member of the GST Alpha class. The 30 kDa protein did not r eact with anti-GST antibodies and was identified as ECI by immunoblott ing and N-terminal-amino-acid-sequencing analyses. The results thus in dicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either a nti-GST antibodies or antibodies against mitochondrial enzymes involve d in the beta-oxidation of fatty acids. However, it exhibited a carbon yl reductase activity with menadione and ubiquinone, and amino acid se quences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl re ductase.