Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000

Abstract

A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of M(r) 91,000 and M(r) 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to ~50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the M(r)-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the M(r)-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new M(r) of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipiated spectral activity and, on Western blots, bound to the M(r)-22,000 polypeptide but not the M(r)-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the M(r)-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.