Electrophoretic isolation of a membrane-bound NADPH oxidase from guinea-pig polymorphonuclear leukocytes.
Electrophoretic isolation of a membrane-bound NADPH oxidase of guinea-pig polymorphonuclear leukocytes was attempted with the O2- -generating membranes of cells unstimulated or stimulated with C3b-zymosan or sodium dodecyl sulfate, and also with the phagosomes isolated from the phorbol myristate acetate-coated latex particle-phagocytosing cells. When these vesicles were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of Triton X-100 and then assayed for NADPH-Nitroblue tetrazolium reducing activity, the activity was detected by the appearance of a single, blue band of the reduced dye on the gel, independent of the source of vesicles. In addition, the enzyme was able to generate O2- and its activity was significantly augmented with the homologous liver microsomal cytochrome b5. Its activity was heat-labile and inactivated by N-ethylmaleimide and p-chloromercuribenzene sulfonate. The enzyme, with an apparent molecular weight of 150 000, in the phagosomes was easily susceptible to limited proteolysis by trypsin and formed an active fragment with a molecular weight of 70 000, accompanying the loss of O2- -generating activity of the vesicles.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003179:Complement C3b / Q000494:pharmacology; D006168:Guinea Pigs; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010587:Phagocytosis; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013481:Superoxides / Q000097:blood; D015054:Zymosan / Q000494:pharmacology
D013481:Superoxides; D012967:Sodium Dodecyl Sulfate; D003179:Complement C3b; D015054:Zymosan; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases