Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat.
In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17alpha-hydroxyprogesterone, androstenedione and testosterone. The transition from C21 to C19 steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17alpha-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-3H]progesterone and [1,2-3H]17alpha-hydroxyprogesterone as substrates. In the testis and male and female duodena, the Km values for progesterone 17alpha-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the Vmax values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the Km values for exogenous 17alpha-hydroxyprogesterone were 525, 675 and 637 nM, whereas the Vmax values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the Km values were calculated with respect to intermediate 17alpha-hydroxyprogesterone formed from progesterone, they were similar to the Km values for 17alpha-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the Vmax values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of Km values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17alpha-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17beta inhibited 17alpha-hydroxylation but not bond scission on intermediate 17alpha-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.
Dalla Valle
L
L
Department of Biology, University of Padova, Italy.
Ramina
A
A
Vianello
S
S
Belvedere
P
P
Colombo
L
L
D000818:Animals; D004386:Duodenum / Q000201:enzymology*; D005260:Female; D007700:Kinetics; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013254:Steroid 17-alpha-Hydroxylase / Q000032:analysis*; D013737:Testis / Q000201:enzymology*
D013254:Steroid 17-alpha-Hydroxylase