Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocytes

• Published in: Biochemical journal Vol. 205; no. 3; pp. 593 - 601
• Main Authors: Wakeyama, H, Takeshige, K, Takayanagi, R, Minakami, S
• Format: Journal Article
• Language: English
• Published: 01.01.1982
• Subjects: leukocytes (polymorphonuclear); NADPH oxidase; pigs; purification
• ISSN: 0264-6021
• DOI: 10.1042/bj2050593

A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mu mol/min per mg of protein with NADPH, and one-fifth of this with NADH, K sub(m) values being similar to those of the vesicle fraction (40 mu M for NADPH and 400 mu M for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH + 2O sub(2) arrow right NADP super(+) + 2O sub(2) super(-) super(.) + H super(+). The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The K sub(m) value for FAD was 0 multiplied by 077 mu M.