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 . However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, 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 . In contrast, disruption of neutrophils by mechanical homogenization resulted in >20% lysosomal rupture and significant plasma membrane proteolysis . Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity .Participation of polymorphonuclear leukocytes (PMN) in host defense against infection is dependent on the ability of this cell to respond to its chemical and physical environment . Although the list of "factors" that stimulate or inhibit PMN function is enormous and ever increasing, the cell's repertoire of responses is essentially limited to three general processes: (a) locomotion in a random or directed fashion (chemotaxis), (b) phagocytosis and killing of ingested material, and (c) secretion of lysosomal constituents . A fourth process, namely protein synthesis, may contribute to the above responses as well as lead to the appearance of new proteins (i .e ., leukocytec pyrogen [1] or serum amyloid A protein [2]) . Each of these major PMN responses is thought to be initiated by an interaction of the appropriate activating factor with the neutrophil plasma membrane . Despite the central role of the plasma membrane in neutrophil function, little information is available on the structural organization and biochemical nature of this cellular component.

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

“…In some experiments, PMN were disrupted by nitrogen cavitation, according to the method described by Klempner et al (21). Briefly, PMN (55 X 106 cells/ml) were suspended in disruption buffer and equilibrated at 400 psi in a cell disruption bomb (Parr Instrument Co., Moline, IL) for 30 min at 4°C.…”

Role of p190RhoGAP in β2 Integrin Regulation of RhoA in Human Neutrophils

“…Briefly, cells were suspended (in 100 mM Tris-HCl (pH 7.5); 5 mM EDTA; 5 mM EGTA; 50 mM NaCl; 5 mM NaF; 1 mM Na 3 VO 4 ; 20 g/ml aprotinin; 1 g/ml each of pepstatin, leupeptin, and antipain; 2.5 mM benzamidine; and 2 mM Pefabloc), placed in a cell disruption bomb at 4°C (27), equilibrated at 1000 lb/in.…”

Identification of a human neutrophil angiotension II-generating protease as cathepsin G.

“…Neutrophils were disrupted by nitrogen cavitation and subcellular fractions were isolated by differential centrifugation (16 (17), ,B-glucuronidase (18), lysozyme (19), ouabain-sensitive Na+K+ adenosine triphosphatase (Na+K+ATPase) (20), and 5'-nucleotidase (21) were assayed as described.…”