Preparation and characterization of plasma membrane vesicles from human polymorphonuclear leukocytes

Abstract

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 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.