Abstract
Soluble cytochrome P-450 from bovine adrenocortical mitochrondria, capable of side chain cleavage, can be incorporated into membranes prepared by dispersion of phospholipids in aqueous buffer when cholate is added to the membrane suspension. In addition, the complete protein side chain cleavae system (i.e. including the ancillary proteins adrenodoxin and adrenodoxin reductase and the substrate cholesterol) can be incorporated into such membranes so that on addition of TPNH, pregnenolone is formed. These components remain in the membrane through gel filtration (which removes almost all the cholate) and sedimentation through sucrose density gradients which separate vesicles without protein and soluble enzyme from the membrane-bound P-450 remains associated with the membrane during and following lysis of vesicles. The vesicles which do not leak [14C]glucose were seen on electron microscopy to show a mean diameter of 350 to 450 A. A number of phospholipids are capable of accomodating P-450 in this manner: mitochondrial lipid extracts, synthetic dipalmitoyl phosphatidylcholine, synthetic dipalmitoyl phosphatidylserine, and egg lecithin, separately or in various combinations. Cholesterol is not necessary for incorporation of the side chain cleavage system. Membrane-bound P-450 shows a Vmax of 28.1 nmol of pregnenolone/min/mg of protein, more than 10 times that of soluble P-450. The spectral properties of the soluble P-450 are altered to become predominantly low spin in the membrane and the enzyme is more stable at 4 degrees C than is soluble p-450.
Highlights
Soluble cytochrome P-450 from bovine adrenocortical mitochondria, capable of side chain cleavage, can be incorporated into membranes prepared by dispersion of phospholipids in aqueous buffer when cholate is added to the membrane suspension
The apparent V, of the membrane-bound enzyme is an order of magnitude higher than that of the soluble enzyme and enzyme activity is more stable at 4°C when the P-450 is associated with a membrane
The term apparent V, serves to emphasize the complexity of the side chain cleavage reaction, the insolubility of the substrate, and the additional step of association between cholesterol and the membrane. In spite of these complications, it can be seen that the enzyme is considerably more active in the membrane than in soluble form (Fig. 5). These findings are observed with membranes reconstituted from a crude extract of mitochondrial lipids and with membranes composed of synthetic phospholipids
Summary
SIDE CHAIN CLEAVAGE SYSTEM FROM PURIFIED (Received for publication, March 10, 1978). California; Irkne, Calif&n& 92717. The spectral properties of the soluble P-450 are altered to become predominantly low spin in the membrane and the enzyme is more stable at 4°C than is soluble P-450 Previous reports from this laboratory described the purification [1] and properties [2,3,4,5,6] of a cytochrome P-450 from bovine adrenocortical mitochondria responsible for catalyzing the side chain cleavage of cholesterol (cholesterol -+ pregnenolone). In studying the properties of the enzyme, it seemed important to investigate the behavior of this cytochrome P-450 in a membrane environment more closely resembling that in which it is normally found For this purpose, we have incorporated the P450 with and without the ancillary proteins (adrenodoxin and adrenodoxin reductase) into membranes formed from mitochondrial phospholipids or from synthetic and naturally occurring phospholipids. This report describes the methods used and the properties of the membrane-bound enzyme
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