Photoaffinity Identification of Colchicine-Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase |
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Authors: | Mark M. Rasenick&dagger ,George L. Wheeler&Dagger ,Mark W. Bitensky&dagger ,Carolyn M. Kosack&dagger ,Rachel L. Malina,Peter J. Stein&dagger § |
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Affiliation: | Department of Neurology, Yale University School of Medicine, New Haven, U.S.A.;Department of Pathology, Yale University School of Medicine, New Haven, U.S.A.;Department of Ophthalmology, Yale University School of Medicine, New Haven, U.S.A.;Department of Chemistry, University of New Haven, West Haven, Connecticut, U.S.A. |
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Abstract: | Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [3H] or [32P](P3-azido-anilido)-P1-5' GTP (AAGTP). When AAGTP-treated membranes were incubated with colchicine or vinblastine and subsequently washed, a single AAGTP-labelled protein of 42 kD was released into the supernatant. About 30% of the total labelled 42-kD protein was released into supernatants from membranes pretreated with colchicine or vinblastine compared with 15% released from control membranes. The amount of adenylate cyclase regulatory subunit (G unit) remaining in these membranes was assessed with reconstitution studies after inactivating the adenylate cyclase catalytic moiety with N-ethylmaleimide (NEM). Forty to fifty percent of functional G units were lost from membranes treated with colchicine prior to washing. This 40-50% loss of functional G unit after colchicine treatment corresponds to the previously observed 42% loss of NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p]-activated adenylate cyclase. Release of the AAGTP-labelled 42-kD protein from colchicine-treated synaptic membranes is double that from lumicolchicine-treated membranes. This colchicine-mediated release of 42-kD protein correlates with a doubling of functional G unit released from synaptic membranes after colchicine treatment. These findings suggest multiple populations of the G unit within the synaptic plasma membrane, some of which may interact with cytoskeletal components. |
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Keywords: | Adenylate cyclase GTP binding protein Cy-toskeleton Microtubule protein Neurotransmitter Receptors Photoaffinity labelling |
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