Membrane splitting by freeze-fracture has been used as a preparative tool for chemical analysis of outer and inner "hal]"-membranes. In a previous report I showed that monolayers of human erythrocytes, bound to cationized glass, fracture nonrandomly, producing membrane fractions substantially enriched in outer or inner "halves." For the present study cells were used in quantities compatible with microanalysis. For quantitation the total amount of membrane present and the fractional enrichment of outer and inner "half"-membranes were determined. Cholesterol was examined by quantitative thin-layer chromatography modified to assay nanogram amounts. Comparison of lipids extracted from intact membranes with lipids from fractured membranes indicated that cholesterol was asymmetrically distributed across the plane of the membrane, more being present on the exterior side than on the interior side.It is widely accepted that the lipids of many biological membranes are arranged in a bilayer (1-3) and that certain lipids of the erythrocyte membrane are asymmetrically distributed across the bilayer (4-8). Evidence for lipid as well as protein and carbohydrate asymmetry has often been derived from chemical labeling and enzymatic degradation of intact erythrocytes compared to isolated membranes or "ghosts" (4-11). Although individual studies often lack rigorous evidence that the label does not penetrate the membrane or that the method does not perturb its structure (12), the bulk of data points strongly to carbohydrate, polypeptide, and phospholipid asymmetry. Still in doubt, however, is the transmembrane-distribution of cholesterol even though it accounts for about 24% of the total lipid of human erythrocytes (13). Although the symmetric distribution of cholesterol in erythrocyte "ghosts" has been suggested (14), most investigators exclude cholesterol from molecular models of the erythrocyte membrane. This uncertainty is in part due to the lack of methods for examining lipids in minimally perturbed membranes.During freeze-fracture membranes are split, as proposed by Branton (15), along an internal plane between terminal methyl-groups in the center of the bilayer. Proteins within this plane appear as particles that partition asymmetrically between the fracture faces, thus providing an electron microscopic marker for the outer or inner "half." Given the means for identification, if one could collect the separated "halves" of fractured membranes, chemical analysis would yield direct information on the transmembrane distribution of components. Although this idea is not new in that preliminary attempts to enrich for "half"-membranes produced by freeze-fracturing have been reported (16)(17)(18). no detailed quantitation of any membrane constituent has been communicated. I recently described a method for producing membrane fractions enriched in outer or inner halves, discussed its electron microscope application, and suggested its potential application to chemical analysis (19). The purpose of this report is to discuss one a...

Membranes of intact erythrocytes bound to polylysine-treated glass fracture nonrandomly when covered with thin copper and frozen. Electron microscopic examination of the glass side reveals extensive areas of outer "half" membrane (B face) and of the copper side, inner "half" membrane (A face). This technique allows the ultrastructural examination of square-centimeter areas of fractured membrane and the chemical analysis of these membrane "halves".

Optical diffraction and image reconstruction can be used to correlate the electron microscope image of the biological membrane with its electron density projection. Such correlation shows that a single purple membrane particle contains 9 to 12 protein molecules--63 to 84 transmembrane alpha helices--a complexity two to ten times greater than that previously suggested for membrane particles.

No abstract

The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to poly sine by its cytoplasmic surface at neutral pH and by its extracellular surface at Iow pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson Purple membrane is a specialized region of the plasma membrane of Halobacterium halobium, a halophilic, photosynthetic microorganism. The lipid content of the purple membrane is similar to, but not identical with, that of the rest of the plasma membrane (1, 2). However, the purple membrane contains only a single protein, bacteriorhodopsin (3), which is arranged in the plane of the membrane in a highly ordered, crystalline array (4). The purple color of bacteriorhodopsin is due to the presence of retinal linked to a lysine residue by a Schiff base, as in the visual pigment rhodopsin. Bacteriorhodopsin differs from rhodopsin in that it is not bleached upon absorption of light. The bacteriorhodopsin molecule instead undergoes a cyclic photoreaction, during which hydrogen ions are translocated from the inside of the cell to the outside medium (5). The special function of bacteriorhodopsin, and of the purple membrane, is therefore to act as a photon-driven proton pump, establishing a concentration gradient of hydrogen ions across the plasma membrane. The chemical potential associated with this pH gradient is then used by the cell for the synthesis of ATP. A recent review summarizes the role of bacteriorhodopsin in providing this photosynthetic capability to H. halobium (6).The molecular structure of bacteriorhodopsin has been determined at about 7-A resolution by high-resolution electron microscopy of unstained, hydrated membranes (7, 8). The protein was found to contain seven a-helices packed parallel to one another and spanning the full thickness of the membrane. The electron microscope structure analysis confirmed many inferences that were made in earlier x-ray diffraction studies of the purple membrane (9, 10). However, the orientation of the three-dimensional model of bacteriorhodopsin, relative to the cytoplasmic and extracellular surfaces of the membrane, was not determined in the initial electron microscope structural study. It is important to know the orientation of the structural model of bacteriorhodopsin relative to the inside and the outside of the c...