Choleragen activation of solubilized adenylate cyclase: Requirement for GTP and protein activator for demonstration of enzymatic activity

Abstract

The requirements for choleragen activation of adenylate cyclase [ATP pyrophosphate-lyase(cyclizing), EC 4.6.1.1.] were investigated by using an enzyme preparation solubilized with Triton X-100 from an extensively washed [rat] brain particulate fraction and partially purified with DEAE-cellulose. Unlike the particulate enzyme this preparation was not activated after incubation with choleragen plus dithiothreitol, ATP and NAD. Addition of the purified protein activator of cyclic nucleotide phosphodiesterase and Ca to the partially purified enzyme increased basal activity somewhat, but choleragen activation was minimal. When cyclase was incubated with GTP, plus the protein activator (and Ca), choleragen markedly increased the activity 3- to 6-fold. When GppNHp [quanylyl imidodiphosphate] and protein activator were incubated with the cyclase prior to assay, activity was elevated but no effect of choleragen was observed. GTP and GppNHp had relatively small effects on cyclase activity in the absence of protein activator or if they were added directly to the assay. Boiled brain supernatant was consistently more effective than protein activator (plus Ca) and GTP, suggesting that other factors are required for maximal cyclase activity after choleragen treatment. The cyclase system is apparently dissociable into several components, all of which may be necessary for optimal regulation of activity. One of these may be the heat-stable Ca dependent protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase that is required along with GTP for demonstration of choleragen activation of partially purified brain adenylate cyclase.