CRITICAL-ASSESSMENT OF THE PRESENCE OF AN NADPH BINDING-SITE ON NEUTROPHIL CYTOCHROME-B558 BY PHOTOAFFINITY AND IMMUNOCHEMICAL LABELING

The presumed NADPH dehydrogenase function of the heterodimeric cytochrome b558 in the neutrophil oxidase complex has been investigated by combined photoaffinity labeling and immunoblot analysis of membrane proteins from bovine neutrophils. The photoaffinity probe was a radiolabeled analog of NADPH, [4-[N-(4-azido-2-nitrophenyl)[H-3]amino]butyryl]NADPH ([H-3]azido-NADPH), and the antibodies were directed against the C-terminal regions of the two subunits of cytochrome b558. Plasma membrane vesicles obtained by differential centrifugation of bovine neutrophil homogenates were routinely used as a source of NADPH oxidase. They were permeabilized by sodium deoxycholate to facilitate the access of NADPH or its azido analog to the totality of the specific binding sites. In the absence of light, azido-NADPH behaved as a competitive inhibitor of NADPH oxidase with a K(i) of 6 muM, and was able to bind to high-affinity specific binding sites with a K(d) of 5-6 muM, indicating a higher affinity of the oxidase for the photoprobe than for the substrate NADPH (K(M) = 30-40 muM). Upon photolabeling, the oxidase was fully inactivated. Following resolution of the membrane proteins by SDS-PAGE, a predominant photolabeled protein band of 80-100 kDa was revealed, which coincided with the large subunit (beta) of cytochrome b558 identified by immunoblot in a parallel gel. The enzymatic deglycosylation of photolabeled neutrophil membranes shifted the masses of both the pbotolabeled band and the immunoreactive beta subunit from 80-100 to 55-65 kDa in accordance with the glycoprotein nature of the beta subunit. Omission of sodium deoxycholate in the photolabeling medium or in the oxidase assay medium resulted in a 3-5-fold decrease of the extent of photolabeling and the rate of O2- production, indicating that, in the preparation of plasma membrane vesicles utilized, the NADPH binding site of the oxidase as well as the site recognized by [H-3]azido-NADPH was predominantly oriented to the inside. With phagosomal vesicles obtained after phagocytosis of phorbol ester coated latex beads, the extent of photolabeling and the oxidase activity were independent of the presence of sodium deoxycholate, which means that both the NADPH binding site of the oxidase and the [H-3]azido-NADPH site were exposed to the outside. Aging of plasma membranes resulted in the gradual loss of both photolabeling of the 80-100-kDa band and O2- production. Taken together, these results indicate that the beta subunit of the plasma membrane-bound cytochrome b558 contains an NADPH binding site that appears to be catalytically competent in the functioning of the oxidase complex. With a bovine neutrophil granule fraction containing 2-3 times more cytochrome b558 per milligram of protein than the plasma membrane fraction, photoirradiation in the presence of [H-3] azido-NADPH resulted in a rather low photolabeling of the 80-100-kDa protein band compared to that of the plasma membrane-enriched fraction.