. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. IM3-99 y = - 1,5652e-2x R"2 = II 20 40 60 SO 100 120 Mil 20 40 60 80 100 Time at 23°C (mm) 120 140 y = - R«= CQ oo 60 120 180 Timeat23°C(min) Figure 9. Association and dissociation of receptor and ligand. (A) Solubilized SQM was combined with n.\l [':5I]-daYFnLRFa and assayed lor specific binding as a function of time. The equilibrium value was estimated to be 1400 cpm from five independent experiments. The inset shows the relation of the difference between the actual an
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. IM3-99 y = - 1,5652e-2x R"2 = II 20 40 60 SO 100 120 Mil 20 40 60 80 100 Time at 23°C (mm) 120 140 y = - R«= CQ oo 60 120 180 Timeat23°C(min) Figure 9. Association and dissociation of receptor and ligand. (A) Solubilized SQM was combined with n.\l [':5I]-daYFnLRFa and assayed lor specific binding as a function of time. The equilibrium value was estimated to be 1400 cpm from five independent experiments. The inset shows the relation of the difference between the actual and equi- librium values of binding as a function of time, under pseudo first-order conditions with [ligand]/[receptor] ~7. (B) Solubilized SQM was in- cubated with radioligand for 3 h. and then 1 ^M acFnLRFamide was added. The amount of bound radioactivity remaining was determined as a function of time in two independent experiments. The initial value was taken as the amount bound just prior to addition of acFnLRFamide. function of ligand concentration produced curved plots whether CHAPS or CHAPSO was employed to solubilize the receptors (Fig. 10). The curved Scatchard plots made it difficult to estimate precisely the affinity and yield of receptors from the solubilization step. Using a nonlinear curve-fitting program (JMP, SAS Institute, Gary, NC), we determined that the data could not be fit to a two-site model. Binding to the solubilized receptors was sensitive to guanine nucleotides as for the membrane-bound receptors (Fig. 11). Again. GTP[-f]S was more potent than GTP. and ATP had no effect over the same range of concen- trations. The IC50s for GTP and GTP[-y]S were n\I and each about 200-fold more potent than ob- served with unsolubilized SQM. The complete inhibition by GTP and GTP[-y]S indicated that all of the active, solubilized receptors were complexed with G-proteins. These results are consistent with a GTP-induced decrease in the affinity of the re
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
