title: Differential sensitivity of basal and opioid-stimulated low Km GTPase to guanine nucleotide analogs.
abstract: In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.
mesh_terms: D000818:Animals; D002460:Cell Line; D004723:Endorphins / Q000494:pharmacology*; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D004723:Endorphins; D006150:Guanine Nucleotides; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases