Isolation and characterization of a constitutive form of rabbit liver microsomal cytochrome P-450.

E.F Johnson

doi:10.1016/s0021-9258(19)86298-1

Abstract

A heretofore unrecognized form of cytochrome P-450 was purified from rabbit liver microsomes with an average yield and purity similar to that of other highly purified forms of cytochrome P-450. Several properties of this cytochrome are contrasted with those of form 2, the major phenobarbital-inducible cytochrome P-450, form 4, the major 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome, and form 6, a cytochrome that is selectively induced in liver microsomes by 2,3,7,8-tetrachlorodibenzo-p-dioxin during the perinatal period. Thes four forms can be distinguished by virtue of their molecular weights as determined using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, by their respective peptide fingerprints, and by the monospecificity of their antisera. Since the enumerated properties are thought to reflect the primary structure of the cytochromes and since the observed differences are extensive, we suggest that these four forms are not derived from a common protein precursor.

Highlights

A heretofore unrecognized form of cytochromP-e450 mapping, and immunological identity
In this report,we describe the isolation and characterization of a hitherto unrecognized form of microsomal cytochrome P450 from rabbit liver
Purification-The procedure developed for the isolation of cytochrome P-450 form 3 is similar to those used in the preparation of the otherforms

Summary

RESULTS

Ml/h is necessary in order to maintain the definition of the eluting cytochrome band. Purification-The procedure developed for the isolation of cytochrome P-450 form 3 is similar to those used in the preparation of the otherforms. The elution of the cytochrome from the resin in the presence of sodium cholate and is eluted when a hydroxylapatite and a subsequent purification step utilizing DEAEcellulose were performed as described earlier [8] When prepared in this manner, form 6 is obtained with a specific content of 14.5to 17.6 nmol of cytochrome P-450/mg of protein and with a yield of 1 to 3% of the microsomal cytochrome P-450 present in the starting material. Minimal molecular weights calculated from the average heme contents of these preparations (19.0 2 1 nmol/mg) yield values of 52,600 f 2,600 which is similar to thevalue obtained using SDS-PAGE

Visible absorption spectra exhibited by the oxidized and

Step volume

Findings

DISCUSSION