Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Abstract

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-45025) from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253,[549][550][551][552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol -mg of protein-', and the protein showed a single spot with an apparent isoelectric point of 7.4 and an M, of 50500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124pmol min-' nmol of cytochrome P-450-1 and towards la-hydroxyvitamin D3 it was 1375 pmol min-' nmol-1. The preparation also catalysed the 25-hydroxylation of 5/J-cholestane-3a,7a-diol at a rate of 1000 pmol min-' nmol ofcytochrome P-450-1 and w -1 hydroxylation of lauric acid at a rate of 200 pmol min-I nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-45025 from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5/3-cholestane-3a,7a-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-45025 was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-45025 from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450. INTRODUCTION25-Hydroxylation is involved in the bioactivation of vitamin D and in bile acid metabolism [1,2]. The 25-hydroxylation of vitamin D3is the first step in the bioactivation of vitamin D3 into its hormonal form, and microsomal 25-hydroxylation is involved in an alternative pathway for bile acid synthesis [2]. So far, no extrahepatic enzyme systems able to catalyse 25-hydroxylation of bile acid intermediates have been described.In a previous study [3], the isolation of a cytochrome P-450 (cytochrome P-45025) from pig kidney microsomes which is active in the 25-hydroxylation of vitamin D3 was described. In the present study the enzyme is further characterized with a monoclonal antibody and by determination of the N-terminal amino acid sequence. The properties of the enzyme are compared with those of liver microsomal 25-hydroxylase. EXPERIMENTAL MaterialsTSK DEAE-5PW and ampholines were from LKB, and Mono S HR 10/10 and Protein A-Sepharose were from Pharmacia. Freund's complete adjuvant was from Difco. Standards for IgG subclass determination were kindly donated by Pharmacia. Preparations and sources for steroid analogues have, been described previously [4]. Enzyme purificationCytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids from pig kidney microsomes was purified to...

Porcine Microsomal Vitamin D3 25-Hydroxylase (CYP2D25)

“…4). The presence of mRNA expressing CYP2D25 in kidney is supported by previous studies reporting the purification from pig kidney of a microsomal enzyme active in 25-hydroxylation of vitamin D 3 (29,30).…”

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

“…The apparently homogeneous jrotein was purified according to the same procedures as de-,cribed for microsomal vitamin D 25-hydroxylase [15] and ,howed the same apparent M, as this enzyme. These findings ogether with the finding that the ratio between la-and 25-iydroxylation was constant during purification of both the mcidney and liver enzymes indicate that microsomal la-hydroxJation is catalyzed by the previously described microsomal jitamin D 25-hydroxylase(s) in pig kidney and liver [14,15]. The ability of purified and recombinantly expressed mitochondrial 'ZYP27 to catalyze both 25-hydroxylation and la-hydroxylaion in vitamin D bioactivation was recently reported [12].…”

“…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.…”

Liver mitochondrial cytochrome P450 CYP27 and recombinant-expressed human CYP27 catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

“…The enzymes were not purified or further characterized. The pig, rabbit, and rat liver mitochondrial sterol 27-hydroxylase (CYP27) is known to catalyze 24-, 25-, and 27-hydroxylations of C27 steroids and vitamin D3 compounds (4)(5)(6)(7)(8)(9)(10)(11)(12). cDNA encoding the human liver CYP27 was isolated by Cali and Russell (13).…”

“…CYP27 from pig (7) and rabbit (15) liver mitochondria was purified as described except that the final hydroxylapatite chromatography step to remove nonionic detergent was omitted. The preparations showed a single protein band upon gel electrophoresis with apparent Mrs of 53,000 (pig) and 52,000 (rabbit).…”