Analysis of membrane halves : Cholesterol ( cell surface / freeze - fracture / membrane splitting / human erythrocytes / cholesterol quantitation )

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 (16-18). 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 approach to harvesting halfmembranes in quantities suitable for microanalysis, the gen173 eral features of transmembrane quantitation, and the specific transmembrane distribution of cholesterol. MATERIALS AND METHODS Erythrocyte Preparation. Human blood was drawn into acid citrate dextrose solution, kept at 0-5°, and washed as described (19). The pellet was diluted with buffer and the number of erythrocytes per ml determined by hemacytometry. Coverglass/Copper Preparation. Coverglasses, no. 1, 24 X 50 mm (Corning Glass Works, Coming, N.Y.), were cleaned and treated with 100 gl of 5 mM polylysine, average molecular weight = 2000 (19). Pure copper sheet, 30 X 57 X 0.508 mm, flattened by pounding, was dipped into 35% nitric acid in distilled water (vol/vol) for 10 sec, then rinsed with glass-distilled water and dried with N2. Erythrocyte Application. Two hundred microliters of buffer containing 1.2 X 109 erythrocytes were applied to each polylysine-treated coverglass. If the droplet failed to flow quickly, edge drying and air/buffer interface effects produced boundaries of lysed cells and the coverglass was discarded. After 10-15 sec the coverglass was washed at 0-5° in three 50-ml changes of buffer (25 sec each change); the back side was wiped dry and free of cells, and then placed cell side against the copper sheet. For binding assays, coverglasses were photographed within 30 sec of preparation; for splitting assays, they were frozen within 10 sec. Binding Assays. Glass-bound erythrocytes were placed against another coverglass, and quadrants were photographed at random with a Zeiss photomicroscope. Coverglass pairs were then scanned with a Cary 14 recording spectrophotometer equipped with scattered transmission accessory (model 1462). For both photography and spectrophotometry, samples were handled rapidly and gently to avoid osmotic or mechanical lysis. Freeze-Fracture. The copper-erythrocyte-glass laminate was frozen in partially solidified Freon-22, -150°, and stored under liquid nitrogen. Fracturing was performed at room pressure with the copper in contact with liquid nitrogen, using a tool carrying a single-edge razor blade to pry the glass from the copper. A diagrammatic representation of the method is shown in Fig. 1. Unfractured controls, either bound to glass or free in microcapillaries, were similarly frozen and stored under liquid nitrogen. Fracturing Assays. Fractured coverglasses were transparent except for patches that appeared pink. Residual pink areas were measured after lipid extraction. Coverglasses were washed with CHCl3:MeOH (2:1), dried with nitrogen, placed in a photographic enlarger, and "printed" at an enlargement of 4.75X. Prints were placed on a light box and areas measured using a compensating polar planimeter (model 620015, Keuffel & Esser Co., Morristown, N.J.). ConProc. Nat. Acad. Sci. USA 73 (1976) BIND FREEZE FRACTURE .-.:.: -.: -.: -:. :; .. 1.copper--w.. . FIG. 1. Micropreparative method for half-membrane enrichment. Cells are bound to a coverglass, placed against a copper plate, and frozen. The glass is pried from the copper to reveal a preferential fracture through the bound membranes. trol coverglasses, split and unsplit, were freeze-dried and similarly enlarged and measured. Electron Microscopy. Coverglasses fractured at room pressure were transferred to a cold stage under liquid nitrogen, covered, and placed in a Varian VE-61 vacuum evaporator. The stage was warmed to about -80° and shadowed with platinum-carbon (20) at an angle of 20 degrees. Replicas were floated off the glass using a 1:1 dilution of 48% HF:distilled water in polystyrene culture dishes, given several distilled water rinses, and examined and photographed with a Siemens 1 electron microscope. Lipid Extractions were conducted under dry N2 at 220 using reagent grade solvents from freshly opened bottles. All glassware was acid-washed. Lipids were extracted according to Bligh and Dyer (21). Because total lipids were in the low microgram range, special care was taken to avoid contamination during handling. The cholesterol standard was a gift of Dr. Wayne Hubbell and was chromatographically pure as determined by thin-layer chromatography with various solvent systems. Thin-Layer Chromatography. Adsorbosil 5 "Prekotes" (Applied Science Laboratories, Inc., State College, Pa.) were washed and activated. Samples were spotted with a holder manufactured to allow critical positioning of a 10-pl Hamilton syringe. Plates were run to 4 cm (origin to front) in CHCI3:acetone (90:10), dried, sprayed with 50% sulfuric acid in distilled water (vol/vol), and charred at 1800 for 30 min (22). Charred spots were scanned with a Mark III CS double-beam recording microdensitometer (Joyce Loebl & Co., Ltd., England). Optical density peaks were integrated electronically or by planimetry.