Isolation and Partial Characterization of the Plasma Membrane of Purified Bovine Neutrophils

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Abstract
1 Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica. 2 Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20–35% of the activity of plasma membrane marker enzymes. 3 Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20–35-fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25-fold enrichment in a Ca2+-dependent ATPase, whose half-maximal reaction velocity is reached at 2.2 × 10-7 M Ca2+. 4 High-resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81000 to 34000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate. 5 Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large-scale purification of the neutrophil plasma membrane, suitable for biochemical studies.