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JBC, Vol. 251, Issue 24, 7929-7939, Dec, 1976
D. A. Haugen and M. J. Coon
Procedures are described for the isolation of two forms of rabbit liver
microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous
state. They are designated by their relative electrophoretic mobilities on
polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2
and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced
animals, has a subunit molecular weight of 48,700. The best preparations
contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme
per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone
but is also present in phenobarbital-induced and untreated animals, was
isolated from all three sources and found to have a subunit molecular
weight of 55,300. The best preparations contain 17nmol of the cytochrome
per mg of protein and 1 molecule of heme per polypeptide chain. Some of the
purified preparations of the cytochromes, although electrophoretically
homogeneous, contain apoenzyme due to heme loss during purification. The
purified proteins contain no detectable NADPH-cytochrome P-450 reductase,
cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of
phospholipid (about 1 molecule per subunit). Amino acid analysis indicated
that P-450LM2 and P-450LM4 are similar in composition, but the latter
protein has about 60 additional residues. The COOH-terminal amino acid of
P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that
of P-450LM4 is lysine. NH2-terminal amino acid residues could not be
detected. Carbohydrate analysis indicated that both cytochromes contain 1
residue of glucosamine and 2 of mannose per polypeptide subunit. The
optical spectra of the oxidized and reduced cytochromes and carbon monoxide
complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and
418 nm characteristic of a low spin hemeprotein, and P450LM4 from
beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes
has maxima at 645 and 394 nm, characteristic of the high spin state. The
spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein
concentrations or upon the addition of detergent (Renex), whereas the
spectrum of P-450LM2 is unaffected by the protein concentration or the
presence of detergent. Electron paramagnetic resonance spectrometry of the
purified cytochromes indicated that oxidized -450lm2 is in the low spin
state, whereas P-450LM4 is largely, but not entirely, in the high spin
state.
properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450
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