Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

John M. Trant

Abstract

This study investigated the steroidogenic enzymes of the elasmobranch interrenal. First, biochemical characteristics of cytochrome P450c21 hydroxylase (P450c21) and 3$\beta$-hydroxysteroid dehydrogenase (3$\beta$-HSD) were determined with incubations of the microsomal fraction of Carcharhinus limbatus (blacktip shark) interrenal tissue. Second, molecular reagents were obtained by isolating and sequencing partial cDNA clones encoding the elasmobranch steroidogenic enzymes cytochrome P450 side chain cleavage (P450scc) and 3$\beta$-HSD from blacktip shark and Daysatis americana (southern stingray) interrenal. Finally, the regulation of Daysatis sabina (Atlantic stingray) interrenal steroidogenesis by peptide hormones such as corticotropin (ACTH) and angiotensin II was investigated by in vitro incubations of interrenal tissue. The blacktip shark forms of P450c21 and 3$\beta$-HSD were similar to mammalian forms in activity. Both enzymes were resistant to the effects of nitrogenous osmolytes and had similar pH optima, but had different temperature optima. Blacktip shark P450c21 displayed a much wider substrate specificity than its mammalian counterparts. The proteins encoded by the isolated cDNAs of southern stingray P450scc, southern stingray 3$\beta$-HSD and blacktip shark 3$\beta$-HSD were only 40-50% identical to other forms of these enzymes. Identity increased in regions known to be highly conserved in these proteins. Northern blot analysis detected single P450scc transcripts in both southern and Atlantic stingray interrenal RNA of approximately the same size (4200 base pairs). Single transcripts were also detected for 3$\beta$-HSD in southern stingray (2400 base pairs) and blacktip shark (1400 base pairs) interrenal RNA. Acute and chronic exposure to ACTH stimulated steroidogenesis in isolated Atlantic stingray interrenal tissue. ACTH induces steroidogenesis through a mechanism which could be blocked by a translational inhibitor (cycloheximide), but not by a transcriptional inhibitor (actinomycin D). Angiotensin II, homologous kidney extract, human chorionic gonadotropin and second messenger analogs and activators had no effect on steroidogenesis. No treatment significantly altered the specific activity of steroidogenic enzymes. Although similar in function and activity, there are differences in the structure and regulation between elasmobranch and mammalian steroidogenic enzymes. Such differences make the elasmobranch interrenal an excellent model system for future comparative studies.

ISBN

9780591133721

Pages

192

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