Studies by Trypsin Digestion in Vesicle Membranes

Purified adrenocortical microsomal P-450~21 was incorporated into vesicle membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine a t a molar ratio of 5 3 : 1. Trypsinolysis of the incorporated P 4 5 0 ~ ~ ~ resulted in the formation of 30-, 2 5 , and 20-kDa fragments. Similar fragment formation was observed by trypsinolysis of bovine adrenocortical microsomes with Western blotting using a n t i P 4 5 0 ~ ~ ~ IgG. In the detergent-solubilized state, trypsin cleaved P-450~21 into very small peptides. Washing of the trypsin-treated vesicles with 500 mM Na2CO3 failed to cause these fragments to separate from membranes. N-Terminal amino acid sequencing of these fragments showed that trypsin cleaved the 267Arg-268Val and 332Arg-333Val bonds of P 4 5 0 ~ ~ ~ . The time course of fragment formation indicated that trypsin cleaved the 267Arg-268Val bond first to produce 30and 25-kDa fragments and subsequently the 332Arg-333Val bond in the 25-kDa fragment to produce the 20-kDa fragment. Neither 2 l-hydroxylase activity, the reduced CO difference spectrum, nor the EPR spectrum of digested P-450~21 differed from those of undigested P 4 5 0 ~ ~ ~ . Heat treatment at 50 OC for 20 min did not cause any decrease in activity of digested P-45Oczl, when the substrate progesterone was present. This high stability toward heat treatment was not observed in the solubilized state. Rotational diffusion experiments on P-450~21 showed that the size of the molecule holding the heme was not changed significantly after digestion. On the basis of these results, P 4 5 0 ~ ~ ~ is concluded to be deeply embedded in the vesicle membranes. Adrenocortical steroid hormones are synthesized from cholesterol by the actions of several cytochrome P-450s and 3fl-hydroxysteroid dehydrogenase A4,A5-isomerase (Takemori & Kominami, 1984). P-450~21~ in the endoplasmic reticulum hydroxylates progesterone and 17a-hydroxyprogesterone at the 21-position (Kominami et al., 1980). These steroids are very hydrophobic and concentrated in biological membranes and may possibly be metabolized in the membrane (Flynn, 1971;Ahmad & Mellors, 1978;Arowsmith & Hadgraft, 1983; Tomida et al., 1978). Thecharacteristicsof membrane-bound P-450~21 have been studied using a system in which purified P-450~21 is incorporated in phospholipid vesicle membranes. Some kinetic studies indicate that P 4 5 0 ~ 2 ~ in vesicle membranes metabolizes substrates concentrated in membranes (Kominami et al., 1986). In a reconstituted electron-transfer system in vesicle membranes composed of P-450~21 and NADPH-cytochrome P-450 reductase, electron transfer has been shown to occur through their random collision in membranes (Kominami et al., 1989). A very recent study with rotational diffusion measurements suggested that P-450~21 was deeply embedded in vesicle membranes (Ohta et al., 1992). Two models have been proposed for the topology of cytochrome P-450 in membranes. Tarr et al. (1983) and Ozols et al. (1985) proposed that the major part of a hepatic microsomal cytochrome P-450 was embedded in membranes, based on the hydropathy profile of the primary structure. * To whom correspondence and reprint request should be addressed. 8 Hiroshima University. University of Tokyo at Komaba. Abstract published in Advance ACS Abstracrs, November 1 , 1993. 1 Abbreviations: P -450~~1 , cytochrome P-450 having steroid 21hydroxylase activity (P-450 21A1); P-45017,~~,cytochrome P-450 having steroid 17a-hydroxylase and C17,CZO-lyase activities (P-450 17A1); P-~~OSCC, cytochrome P-450 having cholesterol desmorase activity (P450 11Al); SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; EPR, electron paramagnetic resonance. 0006-2960/93/0432-12935$04.00/0 Nelson and Strobe1 (1988) showed significant homology in the amino acid sequences of various vertebrate cytochrome P-450s and P-450,,. On the basis of the crystallographic data of the P-450,am structure, they proposed that membranebound cytochrome P-450 was attached to membranes only by the N-terminal region as in the case of NADPH-cytochrome P-450 reductase and NADH-cytochrome bs reductase (Gum & Strobel, 1979; Ozols et al., 1984). The N-terminal anchor model has been supported for cytochrome P-450 by trypsin digestion experiments (Vergeres et al., 1989; Brown & Black, 1989), as well as the location of epitopes for reactive antibodies (De-Lemos-Chiarandini et al., 1987), deletions of DNA sequences around the N-terminal signal sequence (Sakaguchi et al., 1987), and fluorescence energy transfer (Centeno & Gutierrez-Merino, 1992). Several contradicting experimental results have been presented. The importance of hydrophobic amino acids other than theN-terminal region has been reported for P-450~21 and P-45017a~yasc (Chiou et al., 1990; Yanase et al., 1989). A genetically expressed cytochrome P-450 not possessing the N-terminal hydrophobic peptide has been found to bind to membranes (Yabusaki et al., 1988; Clark & Waterman, 1991). A shortened P-450 2E1 lacking amino acids 3-21 was reported to bind to Escherichia coli membranes and to retain the catalytic activity (Larson et al., 1991; Pernecky et al., 1993). MitochondrialP-450sccdoesnot have an N-terminal hydrophobic segment but is an integral membrane protein, for which several regions have been proposed to be responsible for the membrane attachment (Vijayakumar & Salerno, 1992). In this study, the topology of membrane-bound P-450~21 was examined in detail by analysis of tryptic fragments. Trypsin cleaved membrane-bound P-450~21 at two sites, but this did not affect significantly the enzymatic and physicochemical