Photoaffinity Identification of Colchicine‐Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase

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Abstract
Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labeling 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-labeled protein or 42 kD [kilo Daltons] was released into the supernatant. Of the total labeled 42-kD protein .apprx. 30% 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). Of functional G units 40-50% were lost from membranes treated with colchicine prior to washing, which 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. Multiple populations of the G units are found within the synaptic plasma membrane, some of which may interact with cytoskeletal components.