Membrane proteins of intact human erythrocytes were labeled with two fluorescent sulfh dyl reagents. The tagged cells were then subjected to simultaneous liposome treatments for either depletion or enrichment of membrane cholesterol content. Cholesterol depletion, which reduces membrane microviscosity, was followed by a series of fluorescence changes all indicating masking of the membrane proteins. Conversely, increasing the membrane microviscosity by cholesterol enrichment resulted in an appreciable increase of the protein exposure to the aqueous surrounding. These findings strongly suggest that membrane proteins may be vertically displaced upon changes in lipid fluidity, a mechanism that may play a significant role in modulation of antigens and receptors in vivo.It is now widely accepted that the dynamics of membrane proteins play a prime role in most functional characteristics of cells. The main mechanisms whereby the dynamic features of membrane proteins are controlled involve the fluidity of the lipid matrix (1, 2). Thus, the function of membrane carriers, receptors, and even enzymes (3-6) can be markedly affected by changes in the lipid microviscosity. This important cooperation between membrane lipids and proteins is not only expressed in the lateral and rotational mobility of the proteins (2), but also in their degree of exposure to the outer surrounding, as we have recently proposed (7,8). Our results have suggested that membrane proteins may be displaced by changing the lipid microviscosity. Upon increasing the microviscosity, some membrane proteins may be squeezed out and become more exposed to the outer domain, whereas decreasing the microviscosity would result in the converse displacement (7,8). This type of displacement could be one of the mechanisms by which the expression of cellular receptors and antigens is being modulated (9). In the following we present experimental evidence for the above hypothesis. MATERIALS AND METHODS1,5-Dimethylaminonaphthalene-sulfoethyleneimide (dansylaziridine) was obtained from Pierce Chemicals Inc. N-(1-Anilinonaphthalene-4-)maleimide (ANM) was purchased from Teika-Seiyaky Co. Thin-layer chromatography of these compounds established a purity of over 95% and they were therefore used without further purification. N-Methyl-and N-benzylpyridinium salts were prepared by first quaternizing with methyl iodide or benzyl chloride followed by conversion to the perchlorate derivatives. The pyridinium perchlorates were analytically and chromatographically pure.Liposomes of egg lecithin (Lipid Products, England) and of lecithin + cholesterol (Sigma CH-S) in phosphate-buffered saline, pH 7.4, were prepared by sonication at 100 W for 0.5 hr at 00 under N2 with a Branson model 160 sonifier. After sonication the dispersions were centrifuged at 22,000 X g for 0.5 hr to sediment undispersed material. Heparinized human blood samples were collected from normal donors and used within 1 hr after drawing. Erythrocytes were sedimented at 1000 X g and the plasma was removed and kept ...

Growing plant tissue is characterized by a sequence ofmetabolic and hormonal changes which terminate with the death of the tissue (18). This process is referred to as development and senescence of tissue. In the last few years development and senescence of flower petals were studied extensively. Recently attention was focused onto the plasma membranes as a possible location for these processes to occur and be regulated (1,7,15).Because of the cell wall, the plasmalemma of plant cells, unlike the plasmalemma of animal cells, is not accessible to direct observation. This difficulty has been circumvented lately by methods which facilitate the preparation of plant cells free of cell wall, so-called protoplasts, by the action of specific enzymes on the plant tissue (5). We have reported (2) that the microviscosity in the lipid core of the protoplast plasmalemma from rose petals increases markedly with age and that it is much less sensitive to temperature than the microviscosity of mammalian membranes.A similar trend of microviscosity changes upon aging in plant microsomal fractions has been demonstrated recently (16).The following study elaborates on the mechanism of changes in microviscosity of the lipid core in the plasmalemma. The maceration solutions were filtered through a Gelman filter unit with a 0.45-,um membrane into Petri dishes. The lower epidermis of three petals from the second whorl of the flower was peeled off and the remaining parts were placed in the maceration solutions in the dark at 21 ± 1 C for 14 to 17 hr without shaking.After maceration, the crude protoplast suspension was filtered

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.

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