Iodate promoted GTP activation of human fat-cell adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 126.96.36.199] and reversed the usual inhibitory effect which GTP presumably exerts through interaction with a distinct regulatory subunit of the enzyme. GTP (0.1 mm) inhibited adenylate cyclase by 52%, while NaIO3 (0.1 to 5 mm) was minimally stimulatory. GTP stimulation in the presence of IO3- was dependent on the concentrations of both salt and nucleotide with maximal stimulation (up to 2.5-fold times basal) occurring at 0.5-1.0 mm IO3- and 0.1 mm GTP. Iodate salts of Li+, Na+, K+, and Rb+ all enhanced GTP action with similar salt concentration dependence, although the cation species did affect the magnitude of GTP activation. The action of IO3- in promoting GTP stimulation differed in several respects from that of Na+ cation which allows activation by the nucleotide relatively independent of the accompanying anion (M. S. Katz, J. S. Partilla, C. R. Schneyer, M. A. Piñeyro, R. I. Gregerman, 1981, Proc. Nat. Acad. Sci. USA 78, 7417-7421). Effective IO3- concentrations were 100-fold lower than those for Na+.IO3--induced GTP stimulation was not increased by raising the temperature from 30 to 37 °C, in contrast to the temperature dependence of the Na+ effect. Furthermore, unlike Na+, IO3- did not allow stimulation of adenylate cyclase by the nonhydrolyzable GTP analog, 5′-guanylyl-β-γ-imidodiphosphate. Iodate did, however, eliminate inhibition of enzyme by the analog, thereby suggesting that IO3- did not act by inhibiting GTPase activity. Time courses showed that basal adenylate cyclase activity decreased with time but that in the presence of IO3- the initial basal rate of cyclic AMP formation was maintained for at least 10 min. Iodate, when added with GTP, reduced the lag time caused by the nucleotide and produced an increase in enzyme acivity over basal. Vanadate, like IO3-, stimulated adenylate cyclase at millimolar concentrations but had only a minimal effect on the interaction of GTP with the fat-cell enzyme, while molybdate's modulation of the nucleotide effect appeared to be entirely attributable to the accompanying Na+ cation. Our results show for the first time modulation of GTP effect by an anion and suggest that IO3- may be a useful probe of the GTP-binding regulatory protein and its interaction with the adenylate cyclase complex.
ASJC Scopus subject areas
- Molecular Biology