Figure 5

Interaction with Gα _q is not dependent on the ability of Fhit to bind or hydrolyze Ap ₃ A. A, HEK293 cells were co-transfected with either one of pFlag-CMV2 constructs encoding wild-type Fhit, Fhit-Y114F, Fhit-L25W, Fhit-I10W/L25W, Fhit-H96D or pFlag-CMV2 vector and in combination with wild-type Gα_q or constitutively active Gα_q mutant (Gα_qRC). Cell lysates were immunoprecipitated with anti-Flag affinity gel. Numerical values shown above the blot represent the relative intensities of Gα_qRC being co-immunoprecipitated as compared to their corresponding wild-type Gα_q. The band intensity of wild-type Gα_q pulled down by Flag-Fhit was set as 1.0. Data shown represent one of three or more sets of immunoblots; other sets yielded similar results. B, Ap₃A (100 μM) was incubated in the absence (top) or presence of 1 μg GST protein (middle) or 1 μg GST-Fhit protein (bottom) at 37°C for 10 min and then subjected to HPLC analysis. The elution profiles were compared with HPLC elution profiles of nucleotide standards (data not shown) including Ap₃A, AMP, ADP, GTPγS and GDPβS; their relative retention times are marked above the reaction profile. C, Reaction profiles of Ap₃A hydrolysis by 0.5 μg GST-Fhit in the absence (top) or presence of 0.5 μg His-Gα₁₆ which had been pre-incubated (30°C for 30 min) with 100 μM GDPβS (middle) or 100 μM GTPγS (bottom). The amount of GST-Fhit was reduced to 0.5 μg in order to facilitate the detection of possible stimulatory effect of activated His-Gα₁₆. GDPβS was detected as an extra peak (middle) with a retention time of 28.5 min. D, Rate of Ap₃A hydrolysis was analyzed from the profiles shown in B and C; rate of hydrolysis (%) was expressed as a percentage of Ap₃A hydrolyzed during the reaction, i.e. percentage difference between areas under the peaks of Ap₃A before and after the hydrolysis reaction.