Abstract
Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.
Highlights
Superoxide production by phagocytic bloocdells in- to be the terminal component of the system responsible for volves assembly of anactive NADPH oxidase complex donating electronsdirectly to molecular oxygen [22]
Both p47- ponentsinto an active oxidase complex, we have explored phox and p67-phox, used together in the absence of several recombinant DNA expression systems to produce the neutrophil cytosola, re incapable of supportincgell free cytosolic factors for use in cell free oxidase reconstitution production of superoxide, confirming the involvementstudies
In thips aper we report the development of an efficient of other soluble factor(s) in the assembly of an active expression system using recombinant baculoviruses [23, 24]
Summary
Production of Recombinant Baculoviruses BV/p47 and BV/p67Bacuioviruses carrying the cDNAs for human p47-phx andp67-phox were constructed with the transfer vector pVL1392, a derivative of pAC373 containing a mutation within the polyhedrin translation initiation site [24]. Crude recombinant p47-phox preparations were diluted to 2.5 volumes with distilled water and applied to a 10-ml column of CMSepharose Fast Flow (Pharmacia LKB Biotechnology Inc.) equilibrated with 5 mM potassium phosphate, 0.15 mM PMSF, pH 7.0 (CM-Sepharose buffer). Protein containingfractions were identified by total protein assay and immunoblotting with a mixture of anti-p47-phox and anti-p67-phxantibodies as described above The protein in these fractions was precipitated by trichloroacetic acid (10%) or heat (100 "C, 5 min) denaturation, andthe supernatants resulting from a 10-min (16,000 X g) centrifugation were analyzed for flavin fluorescence as described above. Typical reactions (100pl) contained variable amounts of pure recombinant p47phox or p67-phox, 10' cell equivalents of neutrophil cytosol, and 5 X 10' cell equivalents of deoxycholate-solubilized membranes [8], prepared from peripheral blood neutrophils following nitrogen cavitation and differential centrifugation procedures described previously [8].
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