JBC, Vol. 251, Issue 19, 6068-6074, Oct, 1976
Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity
H. P. Wang and T. Kimura
Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by
affinity chromatography using an octylamine-substituted Sepharose column.
The resulting optically clear preparation was stable at -20 degrees for
months. The specific concentration of cytochrome P-450 in the preparation
was about 5 nmol of heme per mg of protein. The preparations were free of
adrenodoxin, adrenodoxin reductase, phospholipids, and other heme
contaminations. Polyacrylamide gel electrophoresis of the purified
cytochrome P-450 preparation treated with sodium dodecyl sulfate and
mercaptoethanol showed a single major band with a molecular weight of about
60,000. The optical absorption spectra of the preparation exhibited Soret
maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex,
respectively. The EPR spectrum showed the characteristic features of the
low spin form of ferric cytochrome P-450 with principal components 1.914,
2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed
two large negative ellipticities at 412 and 350 nm. Fluorescence spectra
showed an excitation maximum at 285 nm and an emission maximum at 305 nm
with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at
285 nm, or an emission maximum at 335 nm when the cytochrome molecule is
excited at 305 nm. After reconstitution with adrenodoxin and its reductase,
this cytochrome P-450 was highly active for cholesterol desmolase with an
NADPH-generating system as electron donor but was not active for steroid
11beta-hydroxylase.
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