M-ACETYLANILIDO-GTP, A NOVEL PHOTOAFFINITY LABEL FOR GTP-BINDING PROTEINS - SYNTHESIS AND APPLICATION

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synth esized and used to identify GTP-binding proteins (G-proteins). This GT P analogue is easily prepared and can be used for photoaffinity labell ing of G-proteins without chromatographic purification. In the presenc e of the beta-adrenergic agonist isoprenaline, it activates turkey ery throcyte adenylate cyclase. This activation persists even when the bet a-adrenergic receptor is subsequently blocked by antagonist, indicatin g that the GTP analogue is resistant to hydrolysis. The apparent K-a f or activation of turkey erytrocyte adenylate cyclase by m-AcAGTP was f ound to be 0.21 mu M, a value similar to that for guanosine 5'-[beta,g amma-imido]triphosphate, m-AcAGTP also effectively inhibited the light -dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [alpha-P-32]m-AcAGTP, followed by immunoprec ipitation of G-protein G(q), identified a labelled protein band with t he mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this prote in was enhanced 9-fold in blue over red illuminated membranes, contain ing metarhodopsin and rhodopsin respectively. Labelling of alpha-subun its of heterotrimeric G-proteins was also demonstrated in turkey eryth rocyte membranes. The ease, of preparation of m-AcAGTP and the chemica l properties of the photoreactive acetophenone make this affinity labe l an important new tool in studies of cellular phenomena mediated by g uanine nucleotide-binding proteins.