Purification and characterization of vitamin D 25-hydroxylase from rat liver mitochondria.
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.
Masumoto
O
O
Department of Biochemistry, Hiroshima University School of Dentistry, Japan.
Ohyama
Y
Y
Okuda
K
K
D000818:Animals; D053493:Cholestanetriol 26-Monooxygenase; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D005260:Female; D006882:Hydroxyapatites; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D006882:Hydroxyapatites; D017886:Durapatite; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase