Control of Enzymes in the Vitamin D Endocrine Complex

Ghazarian, Jacob

Abstract

The principal interest of this study is to continue to define the molecular biochemistry of the kidney enzyme systems associated with the ultimate biological regulation of vitamin D metabolism via the control of biogenesis of the horomonally functional forms of vitamin D. The approach will utilize purified enzyme components, which comprise the vitamin D endocrine system, to assess their functional biochemical activities and their regulation. The studies will monitor variations in existing enzyme molecules, or in rate of enzyme synthesis and degradation. The thrust of the experimental aspects will center on studies concerning the biogenesis of kidney mitochondria and the synthesis of the 1- and 24-hyroxylases. Such studies will include determinations of rates of incorporation of isotopic amino acids into apoproteins and of 59 Fe into heme prosthetic groups. These studies will be extended to test the regulation of the 1- and 24-hydroxylase activities by protein kinase-dependent phosphorylation. Specific incorporation of 32 P into kidney mitrochondrial cytochrome P-450 will be explored. Correlations will be sought between the rates of this incorporation and of the appearanceof 1- or 24-hydroxylase activities in isolated mitochondria. The induction of specific phosphatases as a mechanism for the inactivation of the hydroxylases will be examined. The segment of the kidney nephron that is more directly responsive to prathyroid hormone and to prostaglandins through cyclic mononucleotide and protein kinase activities will be investigated. Kidney tubular and glomerular preparations as published by the Principal Investigator will be used to evaluate the effects of parathyroid hormone and prostaglandins on cyclic mononucleotide metabolism which will be assessed by radioimmunoassay. These hormone effects will be correlated with phosphorylation of specific tubular or glomerular proteins as determined from 32P-containing phosphoproteins separated by slab-gel electrophoresis.. Biotransformations of 25-OH-D in response to cyclic mononucleotides in isolated renal cells will be studied to conclude the molecular action of parathyroid hormone and prostaglandins in the kidney. As little is known about specific enzyme associated processes in vitamin D related skeletal or soft tissue disorders, it can be expected that the detailed enzymological studies of this endocrine system will describe the etiology of the pathogenetic mechanisms and will indicate directions for new therapeutic approaches.