Abstract
Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 10--9 M to 10--10 M) of a highly radioactive (125-I, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 125-I-Labeled insulin, cholera toxin and the plant lictins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Plasma membranes are labeled before homogenization by incubating intact cells briefly at 24 degrees or 4 degrees, or by very brief in situ perfusion of the organ, with the 125-I-Labeled marker. After removing the free 125-I-labeled ligand from the medium by washing (at 4 degrees), the membrane-marker complex remains intact over prolonged (days) periods of time at 4 degrees. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. When desired, the complex can be dissociated deliberately by manipulating experimental conditions such as temperature or by adding specific simple sugars. The most generally suitable marker appears to be WGA. At least in certain tissues (e. g. fat cells) labeling of the plasma membrane with 125-I-WGA and 125-I-isnulin can be performed equally well and selectively in homogenates as in the intact cell. 125-I-Cholera toxin cannot be used in homogenates because of significant binding to nuclei. The use of 125-I-labeled WGA as a specific plasma membrane marker is illustrated in following the course of fractionations, and in quantitating the yield and purity, of plasma membranes from fat cells, lymphocytes, and liver. The results are compared with simultaneous measurements of the plasma membrane enzyme "markers," ATPase, 5-nucleotidase, and basal as well as hormone-stimulated adenylate cyclase activities. The fractionation of liver plasma membranes by aqueous dextran-polyethylene glycol two-phase polymer systems and by conventional differential centrifugation procedures arealso quantitated with the marker, 125I-WGA. Substantial quantities of plasma membrane material are no recovered in the interphase of the two-phase polymer system. Conventional liver fractionation procedures which retain, for further purification, only the readily sedimented pellet (2000 times g, 15 min) discard a very large (at least 70%) questenal hy
Highlights
1251-Labeled insulin, cholera toxin and the plant lectins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes
We have previously reported the use of such 1251-labeled markers for studying insideout plasma membrane vesicles [27]
27.6%, w/v) sucrose density gradients (Fig. 1B) reveals that the lz51-insulin bound to the plasma membrane is readily separated from a very small amount of free 12%nsulin, which is retained in the 8.5y0 isotonic sucrose fractions
Summary
Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 1O-g M to 10F” M) of a highly radioactive (12”1, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 1251-Labeled insulin, cholera toxin and the plant lectins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. At least in certain tissues (e.g. fat cells) labeling of the plasma membrane with l*jI-WGA and *261-insulin can be performed well and selectively in homogenates as in the intact cell. The results are compared with simultaneous measurements of the plasma membrane
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