25-Hydroxyvitamin D3-23-hydroxylase, a renal enzyme in several animal species.

The presence of 23,25-dihydroxyvitamin D3 has been demonstrated in vivo and in vitro by a number of laboratories. In order to evaluate the significance of 23-hydroxylation, renal 23-hydroxylase activity was compared to renal 24-hydroxylase activity in several species before and after treatment with 1,25-dihydroxyvitamin D3. The maximum activity of 23-hydroxylase varied widely among species. Treatment of animals with 1,25-dihydroxyvitamin D3 24 h and again 2 h prior to assay of renal tissue resulted in a 1.7- to 5.2-fold increase in 23-hydroxylase activity and a 3.8- to 20.6-fold increase in 24-hydroxylase activity compared to untreated controls. Maximum activity for both 23- and 24-hydroxylase required the enzyme substrate, 25-hydroxyvitamin D3, and an optimum concentration (30 mM) of an oxidizable substrate such as L-malate to supply the reducing equivalents of NADPH needed. Addition of 10 mumol of magnesium chloride resulted in 19 and 24% increases in activity for 23- and 24-hydroxylase, respectively. L-Malate supported the hydroxylation reactions better than succinate, alpha-ketoglutarate, or pyruvate. The apparent Km of calf renal 23-hydroxylase was 5.7 +/- 1.0 microM and of 24-hydroxylase, 2.0 +/- 0.2 microM. Apparent Km's for 23-hydroxylase varied from a low of 2.7 +/- 0.3 microM in the sheep to a high of 19.1 +/- 0.5 microM in the chick, and for 24-hydroxylase from 0.5 +/- 0.1 microM for the chick to 2.0 +/- 0.2 microM for the calf. Maximum velocity values (Vmax) ranged from 40 +/- 9 pmol/min/g for 23-hydroxylase in the chick to 396 +/- 92 in the calf, and for 24-hydroxylase from 108 +/- 89 pmol/min/g in the chick to 851 +/- 88 in the pig. These results help explain the in vivo metabolite concentrations and the predominance of the C(24)- over C(23)-oxidation pathways. Renal 23-hydroxylase was similar to 24-hydroxylase in that it was inhibited by carbon monoxide (63%), cyanide (51%), and antimycin (67%), required molecular oxygen, and functioned best at physiological pH 7.4. It was also inhibited by p-chloromercuribenzoate (39%), but not by dinitrophenol. The relatively large amount of 23-hydroxylase activity present in renal tissue of the calf and young chicks, dogs, goats, pigs, rats, mice, and sheep suggests a prominent role for this enzyme in vitamin D metabolism.