m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application.
A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that the GTP analogue is resistant to hydrolysis. The apparent Ka for activation of turkey erythrocyte adenylate cyclase by m-AcAGTP was found to be 0.21 microM, a value similar to that for guanosine 5'-[beta,gamma-imido]triphosphate. m-AcAGTP also effectively inhibited the light-dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [alpha-32P]m-AcAGTP, followed by immunoprecipitation of G-protein Gq, identified a labelled protein band with the mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this protein was enhanced 9-fold in blue over red illuminated membranes, containing metarhodopsin and rhodopsin respectively. Labelling of alpha-subunits of heterotrimeric G-proteins was also demonstrated in turkey erythrocyte membranes. The ease of preparation of m-AcAGTP and the chemical properties of the photoreactive acetophenone make this affinity label an important new tool in studies of cellular phenomena mediated by guanine nucleotide-binding proteins.
Zor
T
T
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Halifa
I
I
Kleinhaus
S
S
Chorev
M
M
Selinger
Z
Z
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007163:Immunosorbent Techniques; D007545:Isoproterenol / Q000494:pharmacology; D010777:Photochemistry; D011433:Propranolol / Q000494:pharmacology; D014422:Turkeys / Q000097:blood
C092081:3-acetylanilido-guanosine triphosphate; D000345:Affinity Labels; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D011433:Propranolol; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol