Abstract
The characteristics of cytochrome P450c21 (21 hydroxylase; E.C. 1.14.99.10) were determined in interrenal tissue from blacktip shark (Carcharhinus limbatus). Consistent with other vertebrates, 21‐hydroxylase activity was enriched in the microsomal fraction of the interrenal and was dependent upon a source of reducing equivalents. The reduced form of nicotinamide adenine dinucleotide phosphate supported the highest level of activity. Specific activity increased with increasing temperature (Q~10~ = 2.5) with a maximum at 35°C. At 30°C, maximal 21‐hydroxylase activity reached a plateau around pH 7.5. Elasmobranchs maintain high concentrations of intracellular nitrogenous compounds known to disturb protein function. While urea (400 mM) had no apparent effect upon 21‐hydroxylase activity, trimethylamine oxide (200 mM) inhibited conversion of progesterone to 11‐deoxycorticosterone by 23%. This inhibition was not reversed by urea. Potassium chloride (150 mM) inhibited 21‐hydroxylase activity by 33%. Kinetic parameters of shark 21‐hydroxylase were determined for [^3^H]progesterone (K~m~ = 80 nM; V~max~ = 2.94 nmol/min/mg protein) and [^3^H]17α‐hydroxyprogesterone (K~m~ = 90 nM; V~max~ = 0.89 nmol/min/mg protein). Substituted progestins were used to determine the steric specificity of this enzyme. Of thirteen steroid substrates studied, nine were metabolized, indicating a relatively broad specificity. The addition of trimethylamine oxide did not alter the substrate specificity. Although the blacktip shark homolog of cytochrome P450c21 is similar in some respects to other vertebrate forms, its temperature dependence, preferential use of progesterone and broad substrate specificity are unusual characteristics. © 1995 Wiley‐Liss, Inc.