Phosphorylation of ferredoxin and regulation of renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase activity in vitro.
The kidney is the principal physiologic site of production of biologically active 1,25-dihydroxyvitamin D. The 25-hydroxyvitamin D-1 alpha-hydroxylase (1-OHase) activity found in renal mitochondria is under tight hormonal control. Parathyroid hormone stimulates the renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D in young animals, which is accompanied by dephosphorylation of ferredoxin (Fx), a component of the mitochondrial 1-OHase enzyme complex (Siegel, N., Wongsurawat, N., and Armbrecht, H. J. (1986) J. Biol. Chem. 261, 16998-17003). The present study investigates the capacity of Fx to be phosphorylated in vitro and to modulate the 1-OHase activity of a reconstituted system. Fx was phosphorylated by renal mitochondrial type II protein kinase. Phosphorylation did not alter Fx mobility on sodium dodecyl sulfate gels but did decrease the pI as measured by isoelectric focusing. Amino acid analysis demonstrated that 1 mol of serine and 1 mol of threonine were phosphorylated per mol of Fx. Peptide mapping of phosphorylated Fx was consistent with phosphorylation of serine 88 and threonine 85 or 97. Fx was selectively dephosphorylated by rabbit skeletal muscle protein phosphatase C2 but not C1. Phosphorylation of Fx significantly inhibited the 1-OHase activity of a reconstituted system consisting of Fx reductase, Fx, and renal mitochondrial cytochrome P-450. These findings suggest that phosphorylation/dephosphorylation of Fx may play a role in modulating renal 1,25-dihydroxyvitamin D production.
Nemani
R
R
Department of Pharmacology, Saint Louis University School of Medicine, Missouri 63104.
Ghazarian
J G
JG
Moorthy
B
B
Wongsurawat
N
N
Strong
R
R
Armbrecht
H J
HJ
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism*; D000302:Adrenal Cortex / Q000378:metabolism; D000325:Adrenodoxin / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D005288:Ferredoxins / Q000302:isolation & purification / Q000378:metabolism*; D006706:Homeostasis; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism*
D000596:Amino Acids; D005288:Ferredoxins; D000325:Adrenodoxin; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D011494:Protein Kinases