Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin.
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 Mr 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-gamma-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 alpha-subunit and that the cytosolic fraction of neutrophils contains free alpha-subunits of the same size.
Volpp
B D
BD
Department of Medicine, College of Medicine, University of Iowa 52242.
Nauseef
W M
WM
Clark
R A
RA
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology*; D002918:Chymotrypsin; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol; D037342:Pertussis Toxin; D011092:Polyethylene Glycols; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D011092:Polyethylene Glycols; D017830:Octoxynol; D002772:Cholera Toxin; C010615:Nonidet P-40; D037342:Pertussis Toxin; D002918:Chymotrypsin; D019204:GTP-Binding Proteins