Microsomal and cytosolic fractions of guinea pig hepatocytes contain 100-kilodalton GTP-binding proteins reactive with antisera against alpha subunits of stimulatory and inhibitory heterotrimeric GTP-binding proteins.
Guinea pig hepatocytes fractionated by differential centrifugation into plasma membrane-enriched, microsomal, and cytosolic fractions were examined for their content of alpha and beta subunits of heterotrimeric GTP-binding proteins (G proteins) involved in signal transduction. alpha subunits of stimulatory (Gs) and inhibitory (Gi) proteins were detected by immunoblots with antisera reactive with the carboxyl-terminal decapeptide regions of these proteins. Unexpectedly, antisera (including immunopurified) to the alpha subunit but not the beta subunit reacted with a band of 100-kDa proteins in both the microsomal and cytosolic fractions. The immunoreactive 100-kDa proteins are not substrates for ADP-ribosylation catalyzed by pertussis toxin, cholera toxin, or diptheria toxin. Protease digests of the 100-kDa proteins yielded immunoreactive peptides that are distinctly different from those obtained from protease digests of alpha subunits of heterotrimeric G proteins. The 100-kDa protein(s) reactive with antisera to Gi alpha subunit bind to GTP-agarose but not to ATP-agarose. It is concluded that the immunoreactive 100-kDa proteins in microsomal and cytosolic fractions are structurally distinct G proteins from those linked to receptors in the plasma membrane and other G proteins such as elongation factor 2. Conceivably, the 100-kDa proteins represent a new class of G proteins.
Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
D
D
Udrisar
M
M
Rodbell
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002772:Cholera Toxin / Q000494:pharmacology; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D006168:Guinea Pigs; D007106:Immune Sera; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008970:Molecular Weight; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004167:Diphtheria Toxin; D000939:Epitopes; D007106:Immune Sera; D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins