Structural Studies on Hepatic Microsomal Nadph - Cytochrome P-450 Reductase.

NADPH-cytochrome P-450 reductase, a membrane-bound flavoprotein which contains both FAD and FMN, is a required component of the microsomal cytochrome P-450-containing mixed function oxidase system. This system is composed of the detergent-solubilized reductase, various isozymes of cytochrome P-450, and phospholipid, and serves to effect the oxidation of a wide variety of both physiological and foreign compounds. In the present study, the native rabbit liver reductase (minimal M(,r) 78,000) was shown to have an amphiphilic, two-domain structure. Limited cleavage of the reductase was accomplished with use of a number of endoproteases to produce a large hydrophilic fragment (M(,r) (TURN) 71,000) and a small hydrophobic peptide. The large hydrophilic fragment, which retains both flavins, is approximately 40% more active in the reduction of cytochrome c than is the native enzyme, but has only 3% residual activity toward the physiological acceptor, cytochrome P-450. The small hydrophobic peptide, which was purified in a 77% yield, is highly aggregated in aqueous solution, and is fully soluble only in organic solvents. C-terminal analyses indicated that the native protease-solubilized reductases possessed the same C-terminal sequence: -Trp-(Leu,Val)-Asp-Ser-COOH. N-terminal analysis of the native enzyme showed that a blocking group (later shown to be acetyl) is present; the protease-solubilized forms were readily sequenced. These data prove conclusively that the catalytic domain is the C-terminal polypeptide, while the hydrophobic domain is located in the N-terminal region. After deblocking of the small hydrophobic fragment, sequence analysis revealed an extended hydrophobic region within the peptide. This 17-residue apolar segment with a predicted (beta)-sheet structure is believed to be the site of interaction of the reductase with the phospholipid bilayer. The isolation and sequence of a decapeptide "connecting region" provided the overlap needed to join the hydrophobic and hydrophilic domains, and established a sequence of 69 contiguous residues. Also presented are sequence data on an "adventitious" reductase and the tryptic rat and human reductases, and studies on the liposome-incorporated enzyme, long-term storage stability of the reductase, and a highly efficient method for the separation of PTH-amino acids.PhDBiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158798/1/8214964.pd