Purification and characterization of vitamin D 25-hydroxylase from rat liver mitochondria.

Abstract

Vitamin D 25-hydroxylase was purified from female rat liver mitochondria based on the catalytic enzyme activity. The final preparation of cytochrome P-450 was homogeneous judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,500). The specific content of the purified enzyme was 12 nmol/mg of protein. The absorption spectrum of the purified enzyme showed a peak at 417 nm and that of the dithionite-reduced CO complex at 450 nm, indicating the enzyme belongs to the cytochrome P-450 family. Upon reconstitution with the electron-transferring system of the adrenal system (adrenodoxin and NADPH-adrenodoxin reductase), the enzyme showed a high activity in hydroxylating 1 alpha-hydroxycholecalciferol at position 25 with a turnover number of 3.8 min-1 and Km of 54 microM. The enzyme activity was completely lost when the electron-transferring system was repLaced by that of microsomes (NADPH-cytochrome P-450 reductase purified from rat liver microsomes), confirming that the P-450 enzyme was of the mitochondrial type, not the microsomal type. The omission of cytochrome P-450, adrenodoxin, or NADPH-adrenodoxin reductase resulted in complete loss of enzyme activity. The liver mitochondrial cytochrome P-450 hydroxylates vitamin D3 and 1 alpha-hydroxyvitamin D3 at position 25, but did not show any activity toward xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[a]pyrene. The enzyme activity was not inhibited by aminoglutethimide but slightly inhibited by metyrapone. The enzyme activity was markedly inhibited in an atmosphere of CO:O2:N2, 40:20:40.