Separation and detection of nitroblue tetrazolium-reducing enzymes from human polymorphonuclear leukocytes

doi:10.1016/0022-1759(82)90058-8

Journal of Immunological Methods

Volume 54, Issue 2, 29 October 1982, Pages 175-181

https://doi.org/10.1016/0022-1759(82)90058-8 Get rights and content

Abstract

A technique for detecting the nitroblue tetrazolium-reducing activity of human polymorphonuclear leukocyte enzymes separated on polyacrylamide gels is reported. Slices of gel are incubated in buffer containing nitroblue tetrazolium and either NADPH or NADH. Enzymes capable of reducing nitroblue tetrazolium form a dark band due to the formation of the insoluble formazan. By this technique the soluble fraction of human polymorphonuclear leukocyte sonicates was shown to contain several enzymes capable of reducing nitroblue tetrazolium in the presence of NADPH and NADH.

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Cited by (2)

Electrophoretic isolation of a membrane-bound NADPH oxidase from guinea-pig polymorphonuclear leukocytes
1983, BBA - Biomembranes
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 70000, accompanying the loss of O₂⁻-generating activity of the vesicles.
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Separation and detection of nitroblue tetrazolium-reducing enzymes from human polymorphonuclear leukocytes

Abstract

J. Lab. Clin. Med.

New Eng. J. Med.

Scand. J. Clin. Lab. Invest.