Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

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Abstract
The binding of [35S]GTPγS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 μM. The specific binding was 98%. Different guanine nucleotides were all able to compete with [35S]GTPγS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [35S]GTPγS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [35S]GTPγS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPγS, did not displace [35S]GTPγS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [35S]GTPγS binding was strongly Mg2+-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [35S]GTPγS in the presence of GTP may reflect functional G-protein activity.