Fig. 2.

Ca²⁺ regulation of AC-AplA and AC-AplC. (A) AC-AplA is stimulated by Ca²⁺/CaM. (A1) AC activity in membranes from High Five insect cells infected with baculovirus encoding either AC-AplA or β-gal, assayed in EGTA/Ca²⁺/Mg²⁺ buffers with free Ca²⁺ concentrations ranging from 5 nM to 350 μM, in the presence of 1 μM CaM. Absolute AC activity from a representative experiment (mean ± SD of five replicate assays). Note both AC-AplA and native insect AC (β-gal) display stimulation by Ca²⁺, although the absolute maximal activity of the cells expressing recombinant Aplysia AC is substantially greater. (A2) Calculated activity of AC-AplA as a function of [Ca²⁺]. To calculate the Ca²⁺ stimulation of AC-AplA, for each Ca²⁺ concentration, the absolute activity in membranes from cells infected with β-gal virus was subtracted from the activity in membranes from cells infected with AC-AplA virus. Data represent means ± SEM of AC activity from four separate experiments using membranes from independent populations of High Five cells; each membrane preparation was assayed in five replicates. AC activity at each Ca²⁺ concentration was normalized to the basal activity at 5 nM Ca²⁺. (A3) The stimulation of AC-AplA by Ca²⁺ is CaM-dependent. Data are means ± SEM of data from three experiments on separate populations of cells (F_3,6 = 23.7, P = 0.001). (B) AC-AplC is inhibited by Ca²⁺, independently of CaM. (B1) AC activity in membranes from High Five cells infected with baculovirus encoding either AC-AplC or β-gal, assayed at a range of free Ca²⁺ concentrations from 5 nM to 44 μM, in the presence of 1 μM CaM. Absolute AC activity from a representative experiment (mean ± SD of five replicate assays). (B2) Activity of AC-AplC, calculated as in A2. Note the activity of AC-AplC decreased as the concentration of free Ca²⁺ increased. (B3) The inhibition of AC-AplC by Ca²⁺ is independent of CaM. Data are means ± SEM of data from three experiments on separate populations of cells (F_3,6 = 42.8, P < 0.001). In A1, A2, B1, and B2, some error bars are smaller than symbols. *P < 0.05 for pairwise, posthoc comparisons with 50 nM Ca²⁺ + CaM.