Recently, it was reported that the activity of rabbit P450 1A2 is markedly increased at elevated sodium phosphate concentration. Here, the possible structural change of rabbit P450 1A2 accompanying the NaCl-induced increase in its enzyme activity is investigated by fluorescence spectroscopy, circular dichroism, and absorption spectroscopy. It was found that NaCl increased ␣-helix content and lowered ␤-sheet content of P450 1A2 in the presence as well as in the absence of a phospholipid. Intrinsic fluorescence emissions also increased with increasing NaCl concentration. The low spin iron configuration of P450 1A2 shifted toward the high spin configuration in response to the increased salt concentration. The effect of increased potassium phosphate and NaCl on the P450 1A2 activity was also studied. It was found that the activity increase of rabbit P450 1A2 occurs concomitantly with the conformational change including raised ␣-helix content.Microsomal monooxygenase metabolizes a variety of endogenous and xenobiotic compounds. This enzyme system includes cytochrome P450 (P450) 1 enzymes and NADPH-P450 reductase, which transfers an electron from NADPH to P450. Cytochrome b 5 and NADH-cytochrome b 5 reductase may also contribute to the electron flow (2). Quite recently, Schenkman et al. (3) made an interesting observation that high salt concentration enhances the catalytic activity of rabbit P450 1A2. The P450 1A2 is one of various P450 enzymes that is induced by the polycyclic aromatic hydrocarbon. Since a high concentration of NaCl often stabilizes the proteins by preferential hydration (4), it is possible that this increased activity may accompany structural change of P450 1A2. It seems worthwhile, therefore, to study the molecular properties of structural changes of P450 1A2 in detail. Here, the hydroperoxide system instead of NADPH-P450 reductase and NADPH is used because it may be helpful in understanding the role of P450 1A2 conformation itself.We have studied the spectral change of the P450 brought about by salts and phospholipid in order to see whether the enhanced catalytic activity is related to the structural change of P450 1A2. The relationship between the structure and the activity is studied in detail for the first time in this study. The catalytic reactions by cumene hydroperoxide are studied in order to evaluate the role of the protein conformation in the catalytic function and to see how the environment of the heme is influenced by the conformational change induced by salts. The relationship between conformational changes and activities of the P450 is discussed. EXPERIMENTAL PROCEDURESChemicals-L-␣-Dilauroyl-sn-glycerol-3-phosphocholine (DLPC) and cumene hydroperoxide were from Sigma. 7-Ethoxycoumarin was obtained from Aldrich. 7-Ethoxyresorufin and resorufin were obtained from Molecular Probes, Inc. (Eugene, OR). Other chemicals were of the highest grade commercially available.Enzyme Sources-P450 1A2 was purified from liver microsomes of 5,6-benzoflavone-treated rabbits as described (5). The P450 ...

It has been shown that many proteins, when converted into partially unfolded structures, interact strongly with a lipid bilayer. SecA protein of Escherichia coli is an unusual water-soluble protein which, in the native form, can readily penetrate the membrane and lipid bilayer. In order to see whether the native SecA exhibits partially unfolded characteristics, the stability and solvent accessibility of SecA were studied using various spectroscopic and hydrogen-exchange methods. The results are compared with the reported data for native and molten globule forms of alpha-lactalbumin (alpha-LA), as well as those for apocytochrome c. The exposure of hydrophobic residues of SecA, as monitored by 8-anilinonaphthalene-1-sulfonic acid (ANS) binding, and the extent of amide hydrogen exchange were comparable to those of native alpha-LA. On the other hand, equilibrium unfolding experiments showed that SecA is less stable than native alpha-LA. The results of tryptic digestion and the change of endogenous ATPase activity induced by guanidine hydrochloride were suggestive of the C-terminal half of SecA being more flexible than the rest of the protein. The overall conclusion is that the SecA as a whole has a somewhat open structure due to a relatively unstable C-domain which may facilitate its penetration into a lipid bilayer.

Recently, it was reported that the activity of rabbit P450 1A2 is markedly increased at elevated salt concentration (Yun, C-H., Song, M., Ahn, T., and Kim, H. (1996) J. Biol. Chem. 271, 31312-31316). The activity increase of P450 1A2 coincides with the raised ␣-helix content and decreased ␤-sheet content. The presence of phospholipid magnified this effect. Here, possible structural change of rabbit P450 1A2 accompanying the phospholipid-induced increase in its enzyme activity was investigated by circular dichroism, fluorescence spectroscopy, and absorption spectroscopy. Studies with the reconstituted system supported by cumene hydroperoxide or NADPH showed that the P450 1A2 activities were found to be dependent on the head group and hydrocarbon chain length of phospholipid. Phosphatidylcholines having short hydrocarbon chains with a carbon number of 8 -12 were very efficient for reconstitution of the P450-catalyzed reactions supported by both cumene hydroperoxide and NADPH. It was found that the phospholipid increased the ␣-helix content and lowered the ␤-sheet content of P450. Intrinsic fluorescence intensity is also increased in the presence of phospholipid. The low spin iron configuration of P450 1A2 shifted toward the high spin configuration by most of the phospholipids in the endoplasmic reticulum. Some synthetic phospholipids having short hydrocarbon chains with a carbon number of 10 -12 caused a shift in the spin equilibrium of P450 1A2 toward low spin. The effect of detergents on the activity and conformation of P450 1A2 was also studied. It was found that the addition of detergents to P450 1A2 solution increased the enzyme activity of P450 1A2. Detergents also increased the ␣-helix content and lowered the ␤-sheet content of P450 1A2. Intrinsic fluorescence emissions also increased with the presence of detergents. Octyl glucoside and deoxycholate caused a shift toward high spin. On the other hand, cholate caused a shift toward low spin. It was found that the activity increase of rabbit P450 1A2 coincides with the conformational change including raised ␣-helix content. It is proposed that the interaction with the phospholipid molecules surrounding P450 1A2 in the endoplasmic reticulum is important for a functional conformation of P450 1A2 in a monooxygenase system including NADPH-P450 reductase.

The effect of nonlamellar-prone lipids, diacylglycerol (DG) and phosphatidylethanolamine (PE), on the ATPase activity of SecA was examined. When Escherichia coli PE of the standard vesicles composed of 60 mol% of this lipid and 40 mol% of dioleoylphosphatidylglycerol (DOPG) is gradually replaced with either dioleoylglycerol (DOG) or dioeloyl PE (DOPE), the ATPase activity of SecA present together increased appreciably. On the other hand, when E. coli PE of the standard vesicles was replaced with DOG analogs, the SecA ATPase activity decreased slightly, and when replaced with phosphatidylcholine the decrease in the ATPase activity was more appreciable. When DOPE or E. coli PE was added to PC vesicles, the SecA ATPase activity was enhanced only slightly, suggesting that the hexagonal II structure per se is not

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