The objective of this study is to continue to define the molecular biochemistry of the kidney enzyme systems involved in the biological regulation of vitamin D metabolism. The studies are divided into 4 sections. The first deals with the preparation of hybridomas producing antibodies to the 1-hydroxylase cytochrome P- 450 which we have purified. Mouse lymphocytes will be immunized by in vitro technique because of the technique's minimal requirement for antigen. Screening for hybridomas secreting anti- cytochrome P-450 will be by radioimmunoassay using radio-iodinated antigen as has been used in our laboratory. The second section deals with studies on the genomic regulations of 24-hydroxylase induction by 1,25-(OH)2D3 and elucidation of the genomic relationship between the 1- and 24-hydroxylase. Genomic homology between the two hydroxylases will be studied by the technique of differential hybridization and cloning of complementary DNA by insertion into a vector plasmid and transfecting a host bacterium. Radioactive probes will be synthesized from mRNA and used for screening of clone plasmids by hybrid-select replicating method. Hybrid-arrest cell-free translation analysis will then be used to select the clones chain termination method. The third section deals with the regulation of 1-hydroxylase activity by phosphorylation. Stoichiometry of 32-P incorporation into cytochrome P-450 and ferredoxin will be determined by using renal tubular preparations from which the radioactive components will be immunoprecipitated after exposure of the tubules of PTH. The role of pyrophosphatase in the up-regulation of 1-hydroxylase activity will be investigated to determined whether the synthesis of pyrophosphatase is suppressed by 1,25-(OH)2D3. The fourth section deals with measurement of rates of radiolabeled leucine incorporation into renal mitochondrial cytochrome P-450 and their correlation with hydroxylase activities. The rate-limiting step in the synthesis of the hydroxylases will be decine incorporation into apo-cytochrome P-450. It can be expected that the detailed enzymological studies of this endocrine system will help us describe the etiology of skeletal and soft tissue disorders and will indicate directions for new therapeutic approaches. For example, new insight can be gained into the mechanisms that are inherent in urolithiasis and how such mechanisms give rise to the evaluated serum 1,25-(OH)2D3 levels characteristic of this disease.
