Solubilization and reconstitution of kidney 25-hydroxyvitamin D3 1 α- and 24-hydroxylases from vitamin D-replete pigs

Abstract

Pig kidney mitochondrial 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities have been solubilized with cholate/Emulgen 911 and reconstituted with NADPH, ferredoxin reductase and ferredoxin. All three of these components are required for full catalytic activity of both enzymes. Both products were identified by co-chromatography with authentic metabolites on both normal and reverse-phase h.p.l.c. using solvent systems which were shown to separate 10-oxo-19-nor-25-hydroxyvitamin D3 from 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3]. In addition, periodate treatment of the 24,25-(OH)2-D3 product resulted in complete loss of the product as measured by protein-binding assay. Further purification by p-chloroamphetamine-Sepharose chromatography of a solubilized extract from a pig fed a normal diet increased the specific content of the cytochrome P-450 from 0.019 to 0.239 nmol/mg and the 1 alpha-hydroxylase activity from 4.75 to 268 pmol/h per mg. Activity of the 24-hydroxylase in the crude solubilized extract was 6.3 pmol/h per mg, but was undetectable after partial purification by a p-chloroamphetamine-Sepharose column. However, further fractionation of this material by DEAE-Sepharose chromatography resulted in a further increase in 1 alpha-hydroxylase activity to 430 pmol/h per mg and detection of 24-hydroxylase in a separate fraction at a level of 53 pmol/h per mg. Production of 1,25-(OH)2-D3 was linear with time up to 2 h and was dependent upon ferredoxin concentration as well as cytochrome P-450 concentration in the range of 0-40 nM. In the presence of excess ferredoxin and adequate amounts of cytochrome P-450, 1,25-(OH)2-D3 production was also dependent upon substrate concentrations in the range of 0.25 to 2.5 microM yielding an estimated Km of 1 microM. In the presence of excess substrate and ferredoxin, the catalytic-centre activity of the enzyme was estimated to be 1 h-1.

25-Hydroxyvitamin D3-1 alpha-hydroxylase in porcine hepatic tissue: subcellular localization to both mitochondria and microsomes.

“…2B) (32) or the potential mediation of 25(OH)D3 transport within the mitochondria as occurs to 7-dehydrocholesterol and cholesterol in the liver and adrenal glands, respectively (38,39). At any rate, the Vma,, obtained for the hepatic mitochondrial 25(OH)D3 la-hydroxylase is similar to that observed for the pig and rat renal mitochondrial 25(OH)D3 la-hydroxylases (25,32).However, the Km of this hepatic mitochondrial enzyme is a magnitude higher than that observed for the pig and rat renal la-hydroxylases (25,32) or the reconstituted 25(OH)D3 la-hydroxylase from pig renal mitochondria (40). This difference in enzyme Km may account for why serum 1,25(OH)2D3 begins to increase in anephric humans (35), dogs (35), and pigs (36) as circulating 25(OH)D3 approaches pharmacological levels.…”

“…Every definitive study to date has localized the lahydroxylase to the mitochondria (10,11,40). One exception to this was a study by Paulson et al (44), which described a purported 25(OH)D3 la-hydroxylase to be localized in microsomes of the rat yolk sac.…”

Measurement of vitamin D3 metabolites in smelter workers exposed to lead and cadmium.

“…14 It has been suggested that this second hydroxylation is catalysed by a single enzyme 15 ; however, earlier studies identified two membrane bound multiple enzyme complexes in the mitochondria of cells in the proximal convoluted and straight tubular sections of the nephron. 16 17 Other sites postulated for this hydroxylation include the placenta 18 and the epidermis, especially in the keratinocytes, 19 which are extremely active in psoriatic patients. 20 Absorption of calcium from the gut is increased by 1 ,25(OH) 2 D 3, [21][22][23] whereas it has been shown that 24R,25(OH) 2 D 3 does not aVect this regulation of calcium uptake from the small intestine.…”

A possible role for CYP27 as a major renal mitochondrial 25‐hydroxyvitamin D3 1α‐hydroxylase

“…The lct-hydroxylation takes place primarily in the kidney and the enzyme is known to be a cytochrome P450 located in the inner mitochondrial membrane [1][2][3]. Several efforts have been made to try to characterize an assumed specific mitochondrial let-hydroxylase in kidney -so far without success [3][4][5][6][7]. There are two additional mitochondrial cytochromes P450, CYP24 and CYP27, that are active towards 25-hydroxyvitamin Da [8,9].…”