The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.
The uptake and distribution of ascorbic acid and the effect of extracellular glucose on ascorbic acid transport were investigated in human neutrophils. Freshly isolated neutrophils contained 1.0-1.4 mmol ascorbic acid/L, at least 94% of which was present unbound in the cytosol. Intracellular ascorbic acid was found only in the reduced form. The presence of physiologic amounts of ascorbic acid in the extracellular buffer led to the accumulation of millimolar concentrations of ascorbic acid intracellularly. Accumulation was mediated by a high- and a low-affinity transport activity. The high-affinity transport activity had an apparent Km of 2-5 mumol/L whereas the low-affinity transport activity had an apparent Km of 6-7 mmol/L. Glucose inhibited the uptake and accumulation of ascorbic acid by both transport activities in a concentration-dependent fashion. Glucose-induced inhibition of both ascorbic acid transport activities was completely reversible.
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