Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin

B. D. Volpp, W. M. Nauseef, R. A. Clark

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Neutrophil guanine nucleotide-binding proteins are important components of receptor-mediated cellular responses such as degranulation, chemotaxis, and superoxide production. Because the cytoplasmic granules of neutrophils serve as an intracellular store of receptors and NADPH oxidase components, we investigated the subcellular distribution of substrates for ADP-ribosylation by both pertussis and cholera toxins. Cholera toxin substrates of M(r) 43 and 52 kDa were present only in the plasma membrane fraction. A 39-kDa pertussis toxin substrate was present in the plasma membrane cytosol, and a specific granule-enriched fraction. There were no substrates for either toxin in the primary granules. Quantitative GTP-γ-5 binding was localized predominantly to the plasma membrane fraction (47%), but significant portions were found in the specific granule-enriched fractions (13%) and cytosol (34%) as well. Two-dimensional gel electrophoresis and chymotryptic digests of the pertussis toxin substrate from these three subcellular fractions suggested that they are highly homologous. Triton X-114 phase partitioning was used to investigate the hydrophobicity of the toxin substrates. The pertussis toxin substrates in the plasma membrane and granule fractions behaved like integral membrane proteins, whereas the cytosolic substrate partitioned into both lipophilic and aqueous fractions. ADP-ribosylation converted the substrates to a somewhat less lipophilic form. These data suggest that the specific granules or an organelle of similar density serve as an intracellular store of a G protein with a 39-kDa α-subunit and that the cytosolic fraction of neutrophils contains free α-subunits of the same size.

Original languageEnglish (US)
Pages (from-to)3206-3212
Number of pages7
JournalJournal of Immunology
Volume142
Issue number9
StatePublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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