title: Parathyroid hormone stimulates dephosphorylation of the renoredoxin component of the 25-hydroxyvitamin D3-1 alpha-hydroxylase from rat renal cortex.
abstract: Parathyroid hormone (PTH) stimulates the renal conversion of 25-OH-vitamin D3 to 1,25-(OH)2-vitamin D3 in young animals. There is evidence that PTH acts via cAMP and cAMP-dependent protein kinase, but the identity of the phosphorylated protein(s) is unknown. The present study investigates the possibility that phosphorylation modification of specific components of the renal mitochondrial, cytochrome P-450-linked 25-OH-vitamin D3-1 alpha-hydroxylase is involved in the regulation of 1,25-(OH)2-vitamin D3 production. Mitochondria were isolated from [32P]phosphate-labeled renal cortical slices which had been divided into control and agonist-treated groups. The hydroxylase protein components from the solubilized mitochondria were partially purified using p-chloroamphetamine-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. Phosphorylation was observed only in a protein with an Mr = 12,000 and a pI of 4.2 by autoradiography of the gels. This radiolabeled protein was immunoprecipitated with adrenodoxin antibody. Additionally, the protein in the same Mr region of the polyacrylamide gel reacted with adrenodoxin antibody and co-migrated with bovine adrenodoxin. PTH and forskolin treatment resulted in decreased phosphate incorporation into the protein, whereas A23187 treatment increased the phosphorylation. In parallel experiments, affinity-isolated hydroxylase from control and PTH-treated slices was used to assess in vitro hydroxylase activity using [3H]25-hydroxyvitamin D3 as substrate. The hydroxylase activity derived from PTH-treated tissue was significantly higher than that of control. From these data, it is proposed that renal response to PTH in terms of 25-hydroxyvitamin D3 hydroxylase stimulation involves dephosphorylation of renoredoxin, the ferrodoxin component of this hydroxylase complex.
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism*; D000818:Animals; D005288:Ferredoxins / Q000302:isolation & purification / Q000378:metabolism*; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D011916:Rats, Inbred F344; D013250:Steroid Hydroxylases / Q000378:metabolism*
chemical_list: D005288:Ferredoxins; D010281:Parathyroid Hormone; C050867:renoredoxin; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase