Reciprocal post-translational regulation of renal 1 alpha - and 24-hydroxylases of 25-hydroxyvitamin D sub(3) by phosphorylation of ferredoxin. mRNA directed cell-free synthesis and immunoisolation of ferredoxin

• Published in: Biochemical journal 1990-01, Vol.266 (2), p.385-392
• Main Authors: Mandel, M L, Moorthy, B, Ghazarian, J G
• Format: Article
• Language: English
• Subjects: 1 alpha -hydroxylase; 25-hydroxycholecalciferol
• ISSN: 0264-6021

We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated (poly(A) super(+)) RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha - and 24-hydroxylases of 25-hydroxyvitamin D sub(3). The synthesis of ferredoxin was monitored by determining ( super(35)S)methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha - and 24-hydroxylations of 25-hydroxyvitamin D sub(3) in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha -hydroxylase activity while at the same time it stimulated the 24-hydroxylase. These results indicate that, the stimulatin of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha - and 24-hydroxylases. A model delineating the various aspects of this study is presented.