GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases.
The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.
D000067956:Adenylyl Cyclase Inhibitors; D004912:Erythrocytes / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D013329:Structure-Activity Relationship
D000067956:Adenylyl Cyclase Inhibitors; D006150:Guanine Nucleotides; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins