Summary
5′-Guanylylimidodiphosphate (GMP-PNP) had a biphasic effect on basal adenylate cyclase activity of rat fat cell ghosts, being inhibitory and stimulatory while GTP was mainly inhibitory. At low concentrations of GMP-PNP a transient inhibitory phase preceded the onset of activation. This initial inhibition was overcome by higher concentrations of GMP-PNP, ATP or magnesium.
The stimulatory effects of GMP-PNP were increased by high concentrations of ATP or magnesium, the apparent K m for activation being a function of time. After 5 min of incubation half-maximal activation was obtained at 3 μM GMP-PNP, after 20 min of incubation the K m for GMP-PNP was found to be between 0.1 and 0.3 μM. After 20 min of incubation a 15fold increase of cyclase activity above basal level was observed in the presence of 1 μM GMP-PNP. GTP competitively inhibited the stimulant effect of GMP-PNP. On the other hand, it activated basal activity only under carefully selected conditions.
GMP-PNP and noradrenaline had a synergistic action on cyclase activity. At high substrate concentrations (1 mM ATP) GMP-PNP shifted the apparent K m for activation by noradrenaline from 3 μM to 0.1 μM. At low substrate and high magnesium concentrations 1 μM noradrenaline was unable to stimulate adenylate cyclase. Under these conditions GMP-PNP facilitated the stimulation by the hormone, although GMP-PNP itself inhibited basal activity.
It is suggested that GMP-PNP activates the adenylate cyclase by competing at a common nucleotide binding site with inhibitory agents such as free ATP or GTP. Moreover, the guanyl nucleotide analogue may initiate conformational changes of the enzyme system which facilitate the response to hormones.
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Ebert, R., Schwabe, U. Biphasic effect of 5′-guanylylimidodiphosphate on fat cell adenylate cyclase. Naunyn-Schmiedeberg's Arch. Pharmacol. 286, 297–313 (1974). https://doi.org/10.1007/BF00498312
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DOI: https://doi.org/10.1007/BF00498312