Molecular biology and enzymology of elasmobranch 3 beta-hydroxysteroid dehydrogenase

The conversion of 3 beta-hydroxy-Delta 5 steroids into their respective bio logically active 3-keto-Delta 4 steroid counterparts is catalyzed by a micr osomal enzyme, 3 beta-hydroxysteroid dehydrogenase /5-ene-4-ene isomerase ( 3 beta-HSD). In order to study this enzyme in elasmobranchs, partial cDNA c lones of 3 beta-HSD were isolated from steroidogenic tissue of the southern stingray (Dasyatis americana), spiny dogfish shark (Squalus acanthias), an d the blacktip shark (Carcharhinus limbatus). The deduced amino acid sequen ces of the elasmobranchs were 40-44% identical to mammalian 3 beta-HSD sequ ences, and approximately 50% identical to the rainbow trout (Oncorhynchus m ykiss) form. However, the highly conserved substrate-binding region was 80- 90% identical among the various species. Northern blot analysis of interren al gland RNA indicated a single 2.4 kb transcript for the stingray homolog and a 1.4 kb transcript for the blacktip shark homolog. Blacktip shark 3 be ta-HSD activity was enriched in the microsomal fraction of the interrenal g land and was only detected in the presence of the oxidized forms of nicotin amide adenine dinucleotides. Maximal rates were observed near pH 7.5 and be tween 25 degrees C and 30 degrees C. Urea (400 mM) and trimethylamine oxide (200 mM), normally present in elasmobranch blood, had no effect upon 3 bet a-HSD activity. Kinetic parameters of blacktip shark 3 beta-HSD were determ ined for pregnenolone (K-m = 0.35 +/- 0.06 mu M; V-max = 2.3 +/- 0.11 nmol min(-1) mg protein(-1)) and DHEA (K-m = 0.12 +/- 0.02 mu M; V-max = 1.1 +/- 0.41 nmol min(-1) mg protein(-1)). The kinetic parameters of the stingray 3 beta-HSD for pregnenolone (K-m = 0.13 +/- 0.09 mu M; V-max = 4.0 +/- 0.51 nmol min(-1) mg protein(-1)) and DHEA (K-m = 0.12 +/- 0.08 mu M; V-max = 4 .1 +/- 0.53 nmol min(-1) mg protein(-1)) were similar to those of the black tip shark.