Summary. Previous work has shown that there is a small solvent effect on the electron spin resonance spectra of nitroxide spin labels. The aim of this paper is to develop a semiquantitative treatment of the solvent effects and to use this treatment to estimate the shape of the hydrophobic barrier (i.e., polarity profile) of lipid bilayers.In this semiquantitative treatment of solvent effects, the total effect on the isotropic ]aN coupling constant, AA, is expressed as a sum of terms associated with van der Waals interactions, hydrogen bonding, the charged double layer of phospholipid polar groups and the membrane potential. The magnitude of AA as a function of the electric field is estimated with Hiickel molecular orbital theory and, independently, using the Onsager model. With these relations, estimates of the relative importance of the various solvent effect terms are obtained from accurate ESR measurements on dilute solutions of di-t-butyl nitroxide in thirty-three solvents and calculations of the electric field produced by the charged double layer and the membrane potential.To estimate the shape of the hydrophobic barrier of lipid bilayers, fatty acids and L-~-lecithins with doxyl (i.e., 4',4'-dimethyloxazolidine-N-oxyl) labels bonded to various positions along the lipid chains were diffused into phospholipid vesicles and membranes (from the calf liver microsomal fraction). The shape of the hydrophobic barrier is plotted as a polarity index operationally defined in terms of the AA of the lipid spin labels. The effect of the charged double layers is less important than water penetration except when the spin label is within a few Angstroms of the charged groups. Any effects of a membrane potential on AA are insignificant. A comparison of ESR spectra indicates that significant water penetration into the bilayer occurs in both the pure lipid bilayers and in the membrane preparations.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI‐specific phospholipase C (PI‐PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI‐PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8‐barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI‐PLC in complex with its inhibitor, myo‐inositol, at 2.6 A resolution (R factor = 19.5%). This substrate‐like inhibitor interacts with a number of residues highly conserved among prokaryotic PI‐PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI‐PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.

Cytochrome oxidase (EC 1.9.3.1) isolated from beef-heart mitochondria with an appropriate phospholipid content forms vesicular structures. Lipid-protein interactions in this model membrane system were studied with thelipid spin label, 16-doxylstearic acid. As the phospholipid/protein ratio is varied, two spectral components are observed. At low phospholipid/protein ratios (<0.19 mg of phospholipid per mg of protein) the lipid spin label is highly immobilized. At higher phospholipid content an additional component characteristic of fluid lipid bilayers is evident. By summation of digitalized spectra and subsequent integration it was shown that all composite spectra could be approximated by assuming only two components are present, and that the amount of phospholipid bound to the protein is independent of the extent of the fluid bilayer region. The experimentally determined amount of phospholipid for maximum occupancy of protein-bound sites is about 0.2 mg of phospholipid per 1.0 mg of protein. Calculations show that this ratio is consistent with a single layer of phospholipid surrounding the protein complex. The data are interpreted as evidence for a boundary of immobilized lipid between the hydrophobic protein and adjacent fluid bilayer regions in this membrane model system.There is compelling evidence for the existence of phospholipid bilayers in biological membranes. The results of x-ray diffraction studies (1), differential thermal analysis (2), and spin labeling (3) have all indicated a similarity in lipid behavior between phospholipid bilayers and membranes. Evidence is also accumulating for the existence of globular amphipathic membrane proteins extending into or through the lipid regions of the membrane (4). Assuming this general model to be the case, there must exist a boundary between the fluid bilayer region and the membrane proteins. An interesting question arises as to the properties of the lipid-protein interface. Initially, a well-defined membranous preparation with relatively high protein content and easily characterized functional properties is a good system in which to examine lipid-protein interactions. For this study, we chose cytochrome oxidase (EC 1.9.3.1), which forms a model membrane system with partially-characterized components and functional properties (5, 6). At phospholipid/protein ratios of 0.3-0.7 (w/w), cytochrome oxidase, prepared by the general method of Sun et al. (7), spontaneously forms membranous vesicles (Fig. 1). These vesicular structures are essentially an artificial membrane system, but it is reasonable to suppose that interactions between the cytochrome oxidase protein complex and the phospholipids are meaningful in relation to similar associations in the intact inner mitochondrial membrane. In this paper we report a study of membranous cytochrome oxidase of various phospholipid contents that contains a small concentration of the spin label, 16-doxylstearic acid (8).The properties of the protein-lipid boundary and the bilayer region are examined by electron spi...

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