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Characteristics of plasma membrane isolated from a mouse T lymphoma line: comparison after nitrogen cavitation, shearing, detergent treatment, and microvesiculation
Author(s) -
Culvenor Janetta G.,
Mandel Thomas E.,
Whitelaw Alison,
Ferber Ernst
Publication year - 1982
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240200205
Subject(s) - vesicle , membrane , chemistry , biophysics , differential centrifugation , shearing (physics) , chromatography , biochemistry , biology , materials science , composite material
Plasma membrane was isolated from the mouse T lymphoma cell line WEHI‐22 using four different methods of cell disruption followed by centrifugal fractionation. Disruption by nitrogen cavitation or by shearing with a cell pump produced plasma membrane vesicles of similar buoyant density (1.10 g/ml) and morphological appearance. Few C‐type virus particles were present. Cell disruption with 2% Tween‐40 produced membrane vesicles of similar morphology but lower density (1.09 g/ml). All of the above preparations resulted in vesicles with aggregated intramembranous particles after freeze fracture. Microvesiculation with a sublytic concentration of a lysophosphatidylcholine analog (ET‐12‐H) (0.0032% w/v) produced small membrane vesicles which could be isolated without differential centrifugation. However, these had a slightly higher density than vesicles prepared by cavitation or shearing and were co ntaminated by virus particles. Unlike the other preparations, vesicles prepared with ET‐I2‐H had dispersed intramembranous particles. The enzyme γ‐glutamyl transferase was enriched from 20‐ to 45‐fold in the membrane preparations and proved a suitable plasma membrane marker for these cells whose 5′‐nucleotidase content is very low.