Inhibitory Effects and Spectral Changes on Pig Testicular Cytochrome P-450 (17α-Hydroxylase /Lyase) by 20β-Hydroxy-C21-Steroids

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Abstract
The inhibitory effect of 20.beta.-hydroxy-C21-steroids on oxygenase reaction, .DELTA.16-C19-steroid synthetase, 17.alpha.-hydroxylase and C17,20-lyase activities catalyzed by pig testicular cytochrome P-450 (17.alpha.-hydroxylase/lyase) were studied. .DELTA.16-C19-Steroids synthetase, 17.alpha.-hydroxylase and C17,20-lyase activities were competitively inhibited by 3.beta.,20.beta.-dihydroxypregn-5-ene, 3.beta.,17.alpha.,20.beta.-trihydroxypregn-5-ene, 20.beta.-hydroxypregn-4-en-3-one and 17.alpha.,20.beta.-dihydroxypregn-4-en-3-one, and Ki values for these steroids were 0.11-0.17 .mu.M, 0.36-0.58 .mu.M, 0.68-1.25 .mu.M and 8.11-10.1 .mu.M, respectively. Substrate-induced difference spectra with those steroids were examined. 3.beta.,20.beta.-dihydroxypregn-5-ene and 20.beta.-hydroxypregn-4-en-3-one showed typical type I difference spectra (peak at 386 nm and trough at 420 nm). However, 3.beta.,17.alpha.,20.beta.-trihydroxypregn-5-ene and 17.alpha.,20.beta.-dihydroxypregn-4-en-3-one showed typical reverse type I (or modified type II) difference spectra (peak at 421 nm and trough at 385 nm). From those results of the spectral titration, it was indicated that 3.beta.,20.beta.-dihydroxypregn-5-ene (or 3.beta.,17.alpha.,20.beta.-trihydroxypregn-5-ene) and cytochrome P-450 (17.alpha.-hydroxylase/lyase) formed a one-to-one complex with very high affinity. In addition, the reduction rate of cytochrome P-450 (17.alpha.-hydroxylase/lyase) with Na2S2O4 was decreased by the addition of those 20.beta.-hydroxy-C21-steroids.