2000 Volume 15 Issue 4 Pages 338-348
To identify TA-510 reductase and obtain information on the stereochemical aspects, the reductase activity has been studied in subcellular fractions from 15 human livers, and finally purified using TA-510 enantiomers as substrates.
1. This activity was present in the cytosolic fraction and exhibited strong product stereoselectivity, i.e. both enantiomers were reduced exclusively to trans-alcohol (M1), but not cis-alcohol. Human liver microsomes showed practically no ketone reductase activity.
2. There was also a remarkable variation between the subjects in the substrate stereoselectivity, suggesting that racemic TA-510 was metabolized by at least two reductive enzymes with different stereochemical requirements.
3. The enzyme responsible for the reduction of (+)-TA-510 enantiomer was co-eluted with carbonyl reductase during purification steps, and separated from another enzyme with preference to (-)-TA-510 enantiomer as a substrate. Identity of (+) -TA-510 reductase and carbonyl reductase was also suggested by comparing the kinetic analysis and susceptibility to inhibitors of human liver cytosols and the purified enzyme.
4. (-)-TA-510 reductase was purified to a homogeneous protein and was shown as a monomeric protein with a molecular weight of 36 kDa. Amino acid sequences of five peptides obtained by proteolytic digestion of the purified enzyme were completely identical to the corresponding regions of previously reported 3α-hydroxysteroid/dihydrodiol dehydrogenase.
