INTERACTION OF SOLUBLE BRAIN ADENYLATE-CYCLASE WITH MANGANESE

Adenylate cyclase solubilized from bovine brain with Triton X-100 is inhibited by 250 .mu.M ethylene glycol bis(.beta.-aminoethyl ether)N,N''-tetraacetic acid (EGTA) when the assay is carried out with 6-12 mM Mg2+. A number of divalent cations can prevent the inhibition if they are added with EGTA prior to the adenylate cyclase assay. Reversal of EGTA inhibition once it has been established by incubating the enzyme with the chelator at 30.degree. C for 1-2 min is specific for Mn2+. Addition of 3-4 mM Mn2+ after EGTA inhibition has been established restores the rate to 77% of the control rate without EGTA (measured with the same concentration of Mn2+). Complete reversal cannot be brought about by Ca2+, Ca2+- and Ca-dependent regulator protein or Co2+. The elution position of adenylate cyclase from Sepharose 6B columns is the same whether or not the buffer contains EGTA. After gel filtration in EGTA-containing buffer, all of the control activity (assayed with 3.5 mM Mn2+ and no EGTA) can be recovered if the effluent fractions are assayed with 3.5 mM Mn2+. Addition of Ca2+- and Ca-dependent regulator protein increased the activity but not to that of the control. Removal of free EGTA from the Sepharose 6B column fractions does not reverse the inhibition. Gpp(NH)p (guanosine 5''-(.beta.,.gamma.-imino)triphosphate) activates the enzyme in the presence of EGTA. The activity is lower than that measured without EGTA. EGTA can inhibit adenylate cyclase which has been fully activated by Gpp(NH)p in the absence of the chelator. In both cases, Mn2+ immediately restores the enzymatic rate to that of the fully activated control. The experiments reported suggest that associated Mn2+ is required for full activity of brain adenylate cyclase and that EGTA inhibits the enzyme by chelating Mn2+. The Mn2+ site appears to be independent of the site through which guanine nucleotide activation takes place.