Nucleotide binding properties of cytosolic components required for expression of activity of the superoxide generating NADPH oxidase

Doron Sha'ag*, Edgar Pick

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

The superoxide generating NADPH oxidase was studied in an SDS-activated cell-free system. This system requires the participation of both membranal and cytosolic components. Cytosol derived from elicited peritoneal guinea pig macrophages was fractioned by several nucleotide affinity chromatography procedures. Various such fractionations led to the separation of two distinct factors, both of which are necessary for the activation and/or activity of the superoxide-forming NADPH oxidase. One factor (σ2), bound to octyl, 2′,5′-ADP-, 5′-ATP-, 5′-GTP-agarose and carboxymethyl-Sepharose but did not bind to hexyl, 5′-AMP-, 5′-ADP- and 5′-GDP-agarose. The other factor (σ1) did not bind to any of the above matrices. Subsequent elution of σ2 from 2′,5′-ADP-agarose was effected by ATP, GTP and NADPH but not by NADH. Elution from GTP-agarose was by ATP and GTP but not by NADPH. Elution from ATP-agarose was by ATP, GTP and also, albeit weakly, by NADPH. The above results suggest that σ2 contains a site which recognizes the phosphate group at the ribose 2′ position in adenosine, and a site that recognizes purine nucleotide triphosphates.

Original languageEnglish
Pages (from-to)405-412
Number of pages8
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1037
Issue number3
DOIs
StatePublished - 1 Mar 1990

Funding

FundersFunder number
Council for Tobacco Research U.S.A. Inc.

    Keywords

    • (Macrophage)
    • Cytosolic factor
    • NADPH binding site
    • NADPH oxidase
    • Nucleotide binding
    • Superoxide generation

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