Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450LM.

Detergent-solubilired liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to catalyze electron transfer to cytochrome P-450, whereas the trypsin-solubilized reductase does not. In the present study, treatment of the highly purified detergent-solubilized rabbit liver enzyme (m.w. 77,700) with trypsin was shown to yield a small peptide (m.w. 6,100) as well as the large peptide (m.w. 71,200) which retains the flavin prosthetfc groups. The small peptide, which is hydrophobic in nature as shown by its amino acid composition and solubility properties, is apparently the moiety in the native reductase involved in binding to cytochrome P-450 and to the microsomal membrane. The C-terminalaminoacid sequences of the native reductaseand large fragment are identical [-Trp-(Leu, Val)-Asp-Ser-COOH], thereby indicating that the hydrophobic peptide is located in the N-terminal region of the enzyme. After the liver microsomal hydroxylation system was reconstituted from detergent-solubilized cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence of phospholipid (1,2), we found that trypsin-solubilized "NADPHcytochrome 5 reductase" (3) could not replace the latter enzyme (4,5). More recently,the detergent-solubilized reductase has been obtained in highly purified form from rat (6-9) and rabbit liver microsomes (lo), thereby permitting a structural comparison with the corresponding protease-treated preparations. The present paper describes the isolation and properties of a small hydrophobic peptide derived from the amino-terminal region of the native reductase from rabbit liver microsomes upon treatment with trypsin. The large, soluble peptide obtained in this process has previously been purified and characterized in other laboratories (3,ll). The small peptide appears to be essential in the binding of the native reductase to cytochrome P-450 and to the microsomal membrane. Gum and Strobe1 (12) have recently concluded that a membranebinding domain is present in the rat liver reductase, but the amino acid composition of the domain, calculated by the difference between the detergentand steapsin-solubilized reductase preparations, was not especially hydrophobic.