G-protein alpha-subunits in cytosolic and membranous fractions of human neutrophils.
In plasma membranes of human neutrophils, we identified two major pertussis toxin substrates of 40 kDa Mr with pI values of 5.30 and 5.37. Only the acidic of the two substrates was also present in neutrophil cytosol. Two-dimensional tryptic peptide maps revealed a high degree of homology of cytosolic and particulate substrates. Purified G-protein beta gamma-complex stimulated pertussis toxin-catalyzed [32P]ADP-ribosylation of membranous and cytosolic substrates of neutrophils less than 2-fold and 6-fold, respectively. Hydrodynamic properties of the cytosolic substrate strongly suggested that it exists as a monomer. Purified G-protein beta gamma-complex increased the s20,w value of the cytosolic substrate from 3.3 S to 4.0 S. The GTP analogue, guanosine 5'-O-(3-thiotriphosphate), promoted the release of pertussis toxin substrates from plasma membranes. An antiserum raised against a sequence specific for the Gi2 alpha-subunit reacted with 39-40 kDa proteins in plasma membranes and with an apparently single 40 kDa protein in cytosol. We conclude that neutrophil cytosol contains monomeric Gi2 alpha-subunits which--by interacting with hydrophobic beta gamma-complexes--may reversibly bind to the plasma membrane.
Rudolph
U
U
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Koesling
D
D
Hinsch
K D
KD
Seifert
R
R
Bigalke
M
M
Schultz
G
G
Rosenthal
W
W
D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D009504:Neutrophils / Q000032:analysis* / Q000648:ultrastructure
D019204:GTP-Binding Proteins