Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

Abstract

Neutrophil chemotaxis, phagocytosis and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. Difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. A method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. Neutrophil disruption by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. EM demonstrated that plasma membranes isolated after nitrogen cavitation were sealed vesicles with striking homogeneity.