ISSN:
0021-9541
Schlagwort(e):
Life and Medical Sciences
;
Cell & Developmental Biology
Quelle:
Wiley InterScience Backfile Collection 1832-2000
Thema:
Biologie
,
Medizin
Notizen:
It would be advantageous to prepare models of the neutrophil plasma membrane in order to examine the role of the plasma membrane in transmembrane signal transduction in the human neutrophil and to dissect ligand-receptor interactions and structural changes in the cell surface upon stimulation. A number of investigators have prepared neutrophil membrane vesicles by homogenization, sonication, or centrifugation-techniques that can result in the loss of substantial amounts of surface membrane material, disruption of lysosomes causing proteolysis of membrane proteins, and contamination of the plasma membrane fraction by internal membranes. These limitations have been overcome in the present studies by employing a modification of the method previously developed in this laboratory. Human neutrophils were suspended in a buffer simulating cytoplasmic ionic and osmotic conditions and disrupted by nitrogen cavitation. The resultant cavitate was freed of undisrupted cells and nuclei and then centrifuged through discontinuous isotonic/isoosmotic Percoll gradients, which resolved four fractions: α (intact azurophilic granules), β (intact specific granules), γ (membrane vesicles), and δ (cytosol). The γ fraction was highly enriched in alkaline phosphatase, a marker of the plasma membrane. In addition, this fraction contained 〈5% of the amounts of lysosomes (indicated by lysozyme activity) and nuclei (indicated by DNA content) found in intact cells or in unfractionated cavitate. Furthermore, the γ fraction contained 〈10% of the levels of endoplasmic reticulum, Golgi, mitochondrial, and lysosomal membranes in cells or cavitates, as determined by assays for glucose 6-phosphatase, galactosyl transferase, monoamine oxidase, and Mo1 (CD11b/CD18; Mac-1), respectively. Finally, 75% of the membrane vesicles were sealed, as indicated by assay of ouabain-sensitive (Na+, K+) ATPase activity, and 55% were oriented right-side-out, as determined by exposure of concanavalin A (ConA) receptors and sialic acid residues on the surfaces of the vesicles. These heterogeneous preparations could be enriched for right-side-out vesicles by their selective adherence to ConA-coated plates and subsequent detachment by rinsing the surfaces of the plates with α-methylman-noside. This enrichment protocol did not affect the integrity of the vesicles and resulted in populations in which 〉85% of the vesicles were oriented right-sideout. This procedure thus permits the preparation of sealed, right-side-out membrane vesicles that may be used as valid experimental models of the neutrophil plasma membrane in a variety of functional studies.
Zusätzliches Material:
1 Ill.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1002/jcp.1041410323
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