Cited by 455Neutrophil, Myeloperoxidase and Oxidative MechanismsLipid Membrane Structure and BehaviorEnzyme Structure and Function

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

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Parkos, C A, Allen, R A, Cochrane, C G, Jesaitis, A J·1987·Journal of Clinical Investigation

DOI 10.1172/jci113128PMC PMC442297

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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 Mr 91,000 and Mr 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 approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-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.

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Authors: Parkos, C. A., Allen, R. A., Cochrane, C. G., & Jesaitis, A. J.
Affiliations: Scripps Research Institute (US)
ORCID: Parkos, C A
Jesaitis, A J
Venue: Journal of Clinical Investigation
Publisher: American Society for Clinical Investigation
Published: 1987-09-01
Volume: 80
Issue: 3
Pages: 732-742
ISSN: 0021-9738 (ISSN-L)
DOI: 10.1172/jci113128
PMC: PMC442297
OpenAlex: W2143908574
Cited by: 455
References: 51
Citations by year: 2026: 4 · 2025: 3 · 2024: 5 · 2023: 5 · 2022: 2 · 2021: 4 · 2020: 2 · 2019: 4 · 2018: 4 · 2017: 6 · 2016: 3 · 2015: 5 · 2014: 12 · 2013: 12 · 2012: 6
FWCI: 12.61
Type: journal article
Language: en
Open access: bronze
doi.org
Locations: Journal of Clinical Investigation (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: American Heart Association
U.S. Public Health Service (RR 00833)
National Institute of Allergy and Infectious Diseases (AI17354, R0I AI 22735)
National Center for Research Resources (RR-00833)
Topics: Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords: human granulocytes, polymorphonuclear leukocytes, plasma membrane, N-glycosylation, X-linked chronic granulomatous disease, diisopropylfluorophosphate, wheat germ agglutinin affinity chromatography, leukocyte membrane
MeSH terms: Antibodies, Cell Membrane, Cross-Linking Reagents, Cytochrome b Group, Glycosylation, Granulocytes, Humans, Immunologic Techniques, Molecular Weight, Peptides

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

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