A Demonstration of Erythrocyte Membrane Asymmetry Philip Pederson, Marc Majewski, a n d Peter Lipke Department of Biological Sciences, Hunter College of the City University of New York, 695 Park Ave., New York, NY 10021 Undergraduate students often find i t difficult to visualize the asymmetric distribution of proteins within membranes. Therefore. we have develoned a lab exercise to demonstrate -~~~~ ~, this concept, as part of a three-period analysis of human red hlood cell memhranes in our undergraduate molecular biology course. The experiment consists of isolating erythrocyte memhranes (period I), differential labeling of intact erythrocytes and isolated erythrocyte memhranes with a n impermeable fluorescent dye (period 2), and separation of the proteins by polyacrylamide gel electrophoresis and visualization of the fluorescent bands (period 3). Membrane asymmetry is demonstrntrd hy selertively laheliue the external ~ r u t e i n sof intact red Mood cells and all mem1;rane proteins of a lysed cell preparation with fluorescein isothiocvanate, a nonpermeahle fluorescent dye which covalently h h d s irrr aminn groups. I.aheled proteins are separated hy S1)S-lx1lyucr.vlum1de slab gel eh~ctrophoresisand wsualized on ;l I]\' virwhox at :302 nm. The, Iuheled.. lvsed . crll DreDaration displays fluorescent hands corresponding to the major erythrocyte memhrane proteins, while the labeled intact cell preparation shows only two of these hands, (erythrocyte hand 3 and part of band 4)', leading to the conclusion that the proteins of hands 3 and 4 are the only ones available for labeling on the outer memhrane surface (see figure). ~~
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Materials Reagents 1.3 M and 0.5 M sucrose in 5 mM sodium phosphate buffer pH 7.4
washed cells from outdated hlood (50%hematocrit in isotonic phosphate huffer pH 7.4) with 190 ml of cold lysis buffer. R e ~ e a t e dcentrifugations a t 27,000 X F. and washings with the lysk buffer will broduce a clean membrane pIeparation eventually, h u t we prefer to isolate the memhranes by ultracentrifugation in adiscontinuous sucrose density gradient. Place 4.0 ml of 1.3 M sucrose in the bottom of a 12 ml polyallomer Beckman-Spinco SW-40Ti rotor gradient tuhe (on ice), and carefully overlay with 2.0 ml of 0.5 M sucrose, while avoiding mixing of the layers. Fill the tuhe with lysed memhranes that have been concentrated by centrifugation a t 27,000 X g for 15 min and centrifuge for 90 min a t 90,000 X g. After centrifugation, the membranes can be removed from the interface of the sucrose layers with a pasteur pipet, washed once with lysis huffer to remove sucrose, assayed for protein,3 and stored frozen until needed. Preparation of Intact Whole Cells Wash intact whole cells repeatedly with 0.16 M carbonate buffer pH 8.6, centrifuging at 1000 X g, until the supernatant is only slightly pink or clear, then resuspend to a hematocrit of 50% Fluorescent Labeling Five ml of the suspension of intact cells and 10 mg of isolated membrane protein suspended in 10 rnl of 0.16Mcarhonatehuffer pH 8.6 are each incubated with 5 mg of FITCIcelite at room temperature with occasional gentle shaking by hand. After 45 min unbound FITC/celite is removed by a l-min centrifugation at 1000 rpm in a table-top centrifuge. The supernatant containing the intact whole cell preparation is diluted with 190 ml of cold lysis huffer inorder to lyse the intact cells, and the membranes are isolated by repeated washings with lysis bufferor by sucrose density gradient centrifuga-
Lysis buffer: 5 mM sodium phosphate buffer pH 7.4 on ice Erythrocyte suspension: 50%hematoerit in 110 mM (isotonic)sodium phosphate buffer pH 7.4 0.16 M sodium carbonate huffer DH8.6 Fluorrsmn isothiotyanatr on celw 10q (FITCi (availahle from Calbwrhem or Sigma Chemical CoJ 50%methanol Gel electrophoresis sample huffer: 0.625 M Tris pH 6.8 2.5%sodium dodecyl sulfate (SDS) 50%elvcerol 10 m~ dithiothreitol 0.01%bromphenol blue Equipment Refrigerated centrifuge and accessories (Du Pont/Sorvall RC equivalent Ultracentrifuge (Beekman LS-65B with SW-40Ti rotor assembly and polvallomer gradient tubes-Beckman #331374 or equivaleni) Table-top centrifuge SDS-polyacrylamideslab gel electrophoresis equipment2 UV viewbox Procedure Preparation of Membrane Fragments Lysed red hlood cells can be prepared by diluting 10 ml of -
' Steck, T. L., J. CeN Bioi., 62, (1974).
Studier, W. F., J. Molec. Biol., 79, 237 (1973). Lowry. 0.H., et al., J. Biol. Chem., 193, 265 (1951).
Gel of FlTC labeled erythrocyte membrane proteins. Lanes l and 3 contain labeled rnembranefragrnents: lanes 2and 4contain proteins horn labeled intact cells.
Volume 62
Number 7 July 1985
621
