Figure 2.

Selective HD regulation of PDE. (A) Selectivity of HD synergy with Gα_tGTPγS. The enzymatic activity of PDE (0.5 nM) was determined with the indicated additions to the reactions. The concentrations for HD_i1, HD_q, HD_s, and NHS-HD_t were 2.0 μM, HD_t and HD_g were 1.0 μM, and Gα_tGTPγS was 0.45 μM. Error bars indicate ±SEM derived from values obtained in three independent experiments, with each determination performed in triplicate. PDE activity assay was performed as described in Materials and Methods. The Gα_tGTPγS was prepared from the purified Gα_tGDP as described (47). (B) Selective interaction of the HDs with PDE catalytic core. Reactivity of the HD proteins isolated from different Gα families toward PDE catalytic core was determined with 0.2 nM tPDE in the presence of the indicated HD concentrations. Limited trypsinization to produce tPDE was performed as described in Materials and Methods. Each value is the mean ± SEM of data obtained from three independent experiments. (C) Attenuation of the catalytic core enzyme by HD_t and Gα_tGDP. Attenuation of 0.2 nM tPDE was determined in the presence of the indicated concentrations of either HD_t (•) or Gα_tGDP (○). The activity for each condition is expressed as the fractional inhibition of the activity assayed for the tPDE alone (34.2 ± 1 nmol of cGMP hydrolyzed). The bar presents the fractional inhibition obtained in the presence of 4.5 μM Gα_tGDP and 5 μM HD_t. The error bars are ±SEM for values obtained in three independent experiments. The curves are representative of the saturations from three independent experiments.