TY - JOUR
T1 - Nucleotide binding properties of cytosolic components required for expression of activity of the superoxide generating NADPH oxidase
AU - Sha'ag, Doron
AU - Pick, Edgar
N1 - Funding Information:
This work was supported by grant 1755 from the Council for Tobacco Research U.S.A. Inc., and the David and Natalie Roberts Chair in Immunophar-macology.
PY - 1990/3/1
Y1 - 1990/3/1
N2 - 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.
AB - 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.
KW - (Macrophage)
KW - Cytosolic factor
KW - NADPH binding site
KW - NADPH oxidase
KW - Nucleotide binding
KW - Superoxide generation
UR - http://www.scopus.com/inward/record.url?scp=0025099501&partnerID=8YFLogxK
U2 - 10.1016/0167-4838(90)90044-G
DO - 10.1016/0167-4838(90)90044-G
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AN - SCOPUS:0025099501
SN - 0167-4838
VL - 1037
SP - 405
EP - 412
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 3
ER -