The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils.
Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.
Quinn
M T
MT
Department of Chemistry, Montana State University, Bozeman 59717.
Parkos
C A
CA
Jesaitis
A J
AJ
D000199:Actins / Q000097:blood; D000469:Alkaline Phosphatase / Q000097:blood; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003573:Cytochrome b Group / Q000097:blood; D003902:Detergents; D006801:Humans; D008840:Microfilament Proteins / Q000097:blood; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D015388:Organelles / Q000032:analysis / Q000378:metabolism; D012995:Solubility; D013481:Superoxides / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014909:Wheat Germ Agglutinins / Q000097:blood; D013049:Spectrin
D000199:Actins; D000469:Alkaline Phosphatase; D002352:Carrier Proteins; D003573:Cytochrome b Group; D003902:Detergents; D008840:Microfilament Proteins; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate; D014909:Wheat Germ Agglutinins; D013049:Spectrin