Recent studies have revealed binding of mitochondrial
enoyl-CoA isomerase (ECI) to
S-hexylglutathione-Sepharose, an affinity matrix used for purification of
glutathione transferases (
GSTs), and the
enzyme has been 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-hexylglutathione-
binding proteins of human
hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of
carcinoma and surrounding tissues were applied to an affinity column, and bound 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 two-dimensional gel electrophoresis. The
proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa
proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa
protein exhibited a low but significant activity towards
1-chloro-2,4-dinitrobenzene (4.25 micromol/min per mg of
protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the
GST Alpha class. The 30 kDa
protein did not react with anti-GST
antibodies and was identified as ECI by immunoblotting and N-terminal-
amino-acid-sequencing analyses. The results thus indicated 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 anti-GST
antibodies or
antibodies against mitochondrial
enzymes involved in the beta-oxidation of
fatty acids. However, it exhibited a
carbonyl reductase activity with
menadione and
ubiquinone, and amino acid sequences 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 reductase.