The long term objective of this laboratory is to elucidate the mechanism(s) whereby ACTH regulates the synthesis of enzymes involved in steroidogenesis in the adrenal cortex. Utilizing bovine adrenal cortical (BAC) cells in monolayer culture as a model system, the role of cholesterol, in the form of lipoproteins or in liposomes as well as other steroid substrates, to mediate the induction of synthesis of cytochromes P-450scc, P-45011Beta, P-450C21 and adrenodoxin will be investigated, by immunoprecipitation of these proteins from a cell extract following radiolabeling with [35S]methionine, and also from an in vitro translation system programmed with cellular RNA. Species of cDNA specific for these proteins will be isolated from a cDNA library prepared from BAC poly (A)+ RNA. These cDNA probes will be utilized to determine whether treatment of BAC cells with ACTH, dibutyryl cAMP or cholesterol leads to an increase in the amount of mRNA specific for these enzymes. The nucleotide sequences of these cDNA probes will also be determined and the probes will be utilized to identify the genes which code for these enzymes. Utilizing isolated nuclei as in in vitro transcription system, the effects of ACTH, cyclic AMP analogs, and putative factors mediating the cellular response to ACTH on the rates of transcription of the genes specific for these proteins will be studied. The processing by BAC mitochondria of the precursor forms of P-450scc, P-45011Beta, and adrenodoxin will be further investigated with respect to requirements for energy, specificity of binding to mitochondrial membranes, and specificity of proteolysis. The abiity of mitochondria derived from different organs such as liver, kidney and corpus luteum to process the precursor forms of these proteins will be investigated in order to determine the specificity of the processing reactions. The effects of ACTH and cyclic nucleotide analogs on the activities of other steroidogenic enzymes, for which antibodies are not available, will be examined. These enzymes include: 17Alpha-hydroxylase, 17,20-lyase, and 3Beta-hydroxysteroid dehydrogenase. By means of the proposed experiments it will be possible to take the first steps necessary in the identification and characterization of both the regulatory elements associated with the genes specific for the BAC steroid hydroxylases, and the physiological factors which control their function.
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