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