JBC, Vol. 253, Issue 14, 5042-5047, Jul, 1978
The active form of cytochrome P-45011beta from adrenal cortex mitochondria
M. Ingelman-Sundberg, J. Montelius, J. Rydstrom and J. A. Gustafsson
Cytochrome P-45011beta has been solubilized and partially purified from
bovine adrenal cortex mitochondria by means of chromatography on
Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450
preparations were about 90% pure as judged by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, but had a low specific content
of P-450 (between 1 and 2 nmol of P-450 per mg of protein). In the presence
of purified preparations of adrenodoxin reductase and adrenodoxin, the
partially purified P-450 preparations catalyzed NADPH-supported
11beta-hydroxylation of unconjugated and sulfoconjugated
deoxycorticosterone. In the reconstituted system the hydroxylation of
deoxycorticosterone sulfate proceeded at a much higher rate than in intact
mitochondria, indicating that in the former case interactions between the
hydrophilic substrate and P-450 were facilitated. In the presence of Triton
X-100 the partially purified cytochrome P-45011beta had a Stokes radius of
4.5 nm, a sedimentation coefficient of 3.1 S, and a partial specific volume
of about 0.85 cm3/g. These results indicate that the cytochrome P-45011beta
. Triton X-100 complex had a molecular weight of about 100,000 and that
P-45011beta bound about 1.1 g of Triton X-100 per g of protein. The
P-45011beta . Triton X-100 complex was catalytically active in
hydroxylation reactions supported by NADPH or the hydroxylating agent
ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome
P-45011beta is the active form of the protein.
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