The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex ADP-ribosylation and purification

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Abstract
Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding .alpha.s component and a .beta..cntdot..gamma. component, which modulates the function of .alpha.s. The .alpha.s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5''-[.beta..gamma.-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain .alpha.s, which we have named ''.alpha.sL'', with an apparent Mr of 42,000-45,000, and ''.alpha.sH'' with an apparent Mr of 46,000-51,000 depending on the gel-electrophotetic system used. The .alpha.sL and .alpha.SH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of .alpha.s units. Functionally, however, both forms of .alpha.s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa .alpha.s polypeptides revealed by ADP-ribosylation and a .beta..cntdot..gamma. component. Activation of purified .alpha.s by guanine nucleotides or fluoride can be reversed by addition of purified .beta..cntdot..gamma. component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.