Purification from pig kidney of a microsomal cytochrome P₄₅₀ catalyzing 1α‐hydroxylation of 25‐hydroxyvitamin D₃

Abstract

A cytochrome P,,, catalyzing lcu-hydroxylation of 25-hgdroxyvitamin D, was purified from pig kidney microsomes. '1 he enzyme preparation showed one protein band on gel electrophoresis with apparent M, of 52,500 and a specific cytochrome P, content of 10.7 nmoUmg of protein. The 25-hydroxyvitamin D, lcx-hydroxylase copnrified with the vitamin D, 25-hydroxylase during purification. A cytochrome P,, catalyzing lcu-hydroxylation was purified also from liver microsomes. The apparently homogeneous enzyme showed the same catalytic properties and apparent 194, as the kidney enzyme. The results of the present communication demonstrate the presence in kidney of a previously unknown microsomal lcu-hydroxylase in addition to the assumed specific mitochondrial lcu-hydroxylase. The possibility that microsomal lcu-hydroxylation in pig kidney and liver is catalyzed by the previously described porcine microsomal vitamin D 25-hydroxylase(s) is discussed.Key words: Cytochrome P,,,,; 25-Hydroxyvitamin D, 1 z-hydroxylase; Renal la-hydroxylation; 1 lepatic la-hydroxylation;Microsomal la-hydroxylationThe activation of vitamin D, to its hormonal form, la,25-tiihydroxyvitamin D,, involves an initial 25-hydroxylation in t :le liver. The subsequent la-hydroxylation of 25-hydroxyvit.tmin D, is thought to be catalyzed mainly by a mitochondrial cytochrome P450 in kidney [l-5]. A lot of effort trying to purify and characterize the mitochondrial let-hydroxylase in kidney 1:as been made -so far without success [4,69]. There are no I :ports concerning a possible la-hydroxylation of 25-hyc'roxyvitamin D, in the microsomal fraction of the kidney. 1 .xtrarenal microsomal la-hydroxylase activity towards 25-hycroxyvitamin D, has been reported [5,10,11]. Hollis [l l] found 1 z-hydroxylase activity both in the microsomal and the mitochondrial fractions of pig liver homogenate. None of these i z-hydroxylases was further purified or characterized [l 11. We have recently shown that CYP27, a mitochondrial sterol 27-I-ydroxylase, is responsible for the main la-hydroxylation of Z 5-hydroxyvitamin D, in the mitochondrial fraction of the liver [ .2]. CYP27 is present also in kidney [13]. Another example of \ itamin D hydroxylating cytochrome PdsO that is present in both 1 ver and kidney is the porcine microsomal vitamin D 25-hyc roxylase(s) [14,15]. The primary aim of this report was to study the presence of possible 25-hydroxyvitamin D, la-hyc roxylase in the microsomal fraction of kidney. A kidney I licrosomal cytochrome P450 catalyzing 1 a-hydroxylation was *Corresponding author. Fax: (46) (18) 55-8778. purified to apparent homogeneity. For reasons of comparison also the liver microsomal cytochrome P450 catalyzing la-hydroxylation was purified and studied. 25-hydroxyvitamin D, were obtained from Sigma Chemical Co. and Solvay Duphar BV, respectively. Ketoconazole and 1,2-dianilinoethane were obtained from Division of Janssen Pharmaceutics NV and Sigma Chemical Co., respectively. Hydroxylapatite (Bio-Gel HTP) was from Bio-Rad. Hydroxylapatite was mix...

The Emerging Role of Nutritional Vitamin D in Secondary Hyperparathyroidism in CKD

“…In the kidney, 25D can be taken by the proximal renal tubule with the help of the cell surface receptors megalin and cubulin. The conversion of 25D into its active biological form, 1,25D, is in its majority catalyzed by mitochondrial 1α-hydroxylase and in the minority by microsomal enzymes [38,39]. 1,25D then binds to DBP then delivered to target tissue to regulate a wide variety of VDR genes.…”