Reciprocal post-translational regulation of renal 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin.
We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] 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 D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, 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 D3 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. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation 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.
Mandel
M L
ML
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Moorthy
B
B
Ghazarian
J G
JG
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002117:Calcitriol / Q000378:metabolism*; D002645:Chickens; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System; D005288:Ferredoxins / Q000378:metabolism*; D015870:Gene Expression; D007668:Kidney / Q000201:enzymology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013250:Steroid Hydroxylases / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D005288:Ferredoxins; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002117:Calcitriol