AbstractIn membranes derived from NG108–15 cells, the opioid peptide [D‐Ala2,D‐Leu5]enkephaIin (DADLE) stimulates a lowKmGTPase. The nucleotide analogs guanosine 5′‐O‐(2‐thio)diphosphate (GDPβS), guanosine 5′‐(β,γ‐imi‐do)triphosphate [Gpp(NH)p] and guanosine 5′‐O‐(3‐thio)‐triphosphate (GTPγS) inhibit the basal enzymatic activity with the order of potency GTPγS>Gpp (NH)p>GDPβS. In the presence of DADLE, the inhibition isotherms of GDPβS 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 IC50of GTPγS for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTPγS produce a concentration‐dependent increase in theKm(app)of GTPase. without affecting itsVmax, indicating a competitive inhlbi‐tion. However, the replots ofKm(app)versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibi‐tion. Both Gpp(NH)p and GTPγS, but not GTP, induce an increase in the EC50of DADLE for stimulating GTPase These findings indicate that the basal and the opioid‐stimu‐lated lowKmGTPase differ in their respective sens