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Photoaffinity Identification of Colchicine‐Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase
Author(s) -
Rasenick Mark M.,
Wheeler George L.,
Bitensky Mark W.,
Kosack Carolyn M.,
Malina Rachel L.,
Stein Peter J.
Publication year - 1984
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1984.tb05407.x
Subject(s) - cyclase , colchicine , adenylate kinase , gtp' , membrane , biochemistry , photoaffinity labeling , chemistry , g protein , biology , binding site , receptor , enzyme , genetics
Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [ 3 H] or [ 32 P](P 3 ‐azido‐anilido)‐P 1 ‐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.