Lymphocyte Plasma Membranes: Analysis of Proteins and Glycoproteins by SDS-Gel Electrophoresis

Lopes, J. D.; Nachbar, Martin S.; Zucker‐Franklin, Dorothea; Silber, Robert

doi:10.1182/blood.v41.1.131.131

Cited by 41 publications

(10 citation statements)

References 26 publications

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“…46000; different membrane preparations had virtually identical patterns (Plate 1). A comparable range of molecular weights, as well as a major polypeptide chain of about 46000daltons, has been found in plasma membranes from kidney and liver cells (Neville & Glossmann, 1971), rat thymocytes (Zimmerman, 1974), human lymphocytes (Ragland et al, 1973;Lopes et al, 1973) and HeLa cells (Atkinson & Summers, 1971). The polyacrylamide-gel pattern was the same whether the membrane sample was fully reduced with 2-mercaptoethanol or not reduced, indicating that most of the membrane polypeptides are not cross-linked by intermolecular disulpaide bonds.…”

Section: Phospholipid Analysesmentioning

confidence: 76%

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes

Chavin¹,

Sm²,

Holliman³

1975

Biochemical Journal

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Plasma membranes from pig mesenteric lymph-node lymphocytes contain a large number of polypeptide chains ranging in molecular weight from 20,000 to greater than 470,000, with a major component of 46,000. There are approx. 12-15 glycoproteins. The membranes contain immunoglobulin G, which comprises 0.6% of the total protein. Immunoglobulin M is also detected, but has not been accurately quantified for technical reasons. Possible origins of the membrane-associated immunoglobulin are briefly discussed. It is concluded that the immunoglobulin is probably associated with the plasma membrane in the intact cell.

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Section: Phospholipid Analysesmentioning

confidence: 76%

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes

Chavin¹,

Sm²,

Holliman³

1975

Biochemical Journal

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“…Yet a satisfactory yield of plasma membrane requires that all cells be disrupted, while nuclei stay undamaged. Homogenization of hypotonically shocked ceils has been used (2,3,14,25,28,31,52), but the efficiency of the procedure has been evaluated in only one case where it was 25% (52). Nitrogen cavitation gave apparently better results (18,39,45), but the efficiency of breakage was not documented and the gelatinous character of the nuclear pellet indicated the lysis of nuclei (45).…”

Section: Discussionmentioning

confidence: 99%

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

Monneron¹,

d’Alayer²

1978

The Journal of Cell Biology

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The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, 7-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase.Plasma membranes and light nuclear membranes appeared as pure unitmembrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.KEY WORDS lymphocyte plasma membrane nuclear membrane membrane marker enzymes ultrastructure subcellular fractionation T cells play a central role in immunological processes. They are responsible for the recognition of many antigens and respond to them by activating surface signals and releasing soluble factors which trigger B cells in various ways (7,30). They are also the effectors of cellular immunity. Such functions are mediated by the plasma membrane. Work is in progress in several laboratories to characterize the receptors of antigens, now known as the products of the Ir genes, and the surface signals, presumably related to the molecules responsible for histocompatibility (38). To study such molecules, it appears likely that they must be maintained in their structural context. Therefore, it is essential to obtain purified plasma membranes of T cells, and of their precursors, the thymocytes.T and B cells, despite their profound biological differences, are morphologically indistinguishable (1, 5). Thymocytes, the precursors of T cells, resemble very much circulating lymphocytes when isolated in a culture medium. Therefore, methods J. C~LL BIOLOgy 9 The Rockefeller University Press 9 0021-9525/78

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“…In trying to compare our SDS electrophoresis analysis with that of others (9,10,13,32,47,49,52,54), we sometimes encountered difficulties due to discrepancies in apparent molecular weight estimations, especially in the high range. However, certain assumptions being made, the pattern that we obtained had some features in common with some of the published analyses (9,10,32,47,49,54). Our plasma membranes were characterized by a number of high molecular weight glycoproteins.…”

Section: Discussionmentioning

confidence: 99%

Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition

Monneron¹,

d’Alayer²

1978

The Journal of Cell Biology

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Thymocyte plasma and nuclear membranes obtained by the procedure described in the accompanying paper were analyzed for their biochemical composition. Plasma membranes were very rich in phospholipid, cholesterol, sialic acid; they did not contain nucleic acids. In comparison, nuclear membranes had a lower phospholipid to protein ratio and contained much less sialic acid and cholesterol. 50% of the cellular cholesterol and of the membrane-bound sialic acid were found in the plasma membranes, 14% in the nuclear membranes. Live cells were labeled with lalI, and the acid-insoluble radioactivity was followed in the subfractions. A good correlation with the distribution and enrichment of plasma membrane marker-enzymes was obtained. Label enrichment was about 50-fold in the two lightest of the three plasma membrane fractions. 60% of the label was contained in the plasma membranes, only 4% in the nuclear membranes, Crosscontamination of these two types of membranes was thus negligible. Sodium dodecyl sulfate-gel electrophoresis revealed three different patterns specific for, respectively, plasma membranes, the microsomal-mitochondrial fraction, and nuclear membranes. Each pattern was characterized by a set of proteins and glycoproteins, among which high molecular weight glycoproteins could be considered as marker-proteins of, respectively, 280,000,260,000, and 230,000 daltons. 131I-labeling of live cells tagged with a very high specific activity three glycoproteins of tool wt 280,000, 200,000, and 135,000 daltons. Nuclear membranes prepared from labeled isolated nuclei had a set of labeled proteins completely different from plasma membranes.KEY WORDS lymphocyte plasma membrane 9 nuclear membrane 9 biochemical composition glycoproteins radioiodinationIn the preceding paper, we described a procedure for thymocyte subfractionation, yielding within a short time and with a high recovery, plasma membranes on the one hand, and nuclear membranes on the other (40). On the basis of ultrastructural and enzymatic studies, these membranes appeared to be well separated from mitochondria, microsomes, and other organelles. The biochemical characterization of the subcellular fractions is reported in this paper. The distribution 232 J. CELL BIOLOGy 9 The Rockefeller University Press 9

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Lymphocyte Plasma Membranes: Analysis of Proteins and Glycoproteins by SDS-Gel Electrophoresis

Cited by 41 publications

References 26 publications

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition

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