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, gamma-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 unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.
Article| April 01 1978
Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure
Online Issn: 1540-8140
Print Issn: 0021-9525
J Cell Biol (1978) 77 (1): 211–231.
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A Monneron, J d'Alayer; Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure. J Cell Biol 1 April 1978; 77 (1): 211–231. doi: https://doi.org/10.1083/jcb.77.1.211
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