Receptor fragment approach to the binding between CCK8 peptide and cholecystokinin receptors: A fluorescence study on type B receptor fragment CCK_B‐R (352–379)

Abstract

Fluorescence titrations in a membrane mimetic solvent system allowed us to estimate that the dissociation constant of the bimolecular complex between CCK8 peptide and cholecystokinin type B receptor fragment CCK(B)-R (352-379) is in the micromolar range. When considered in the context of the full receptor/ligand model, these experiments demonstrate that the receptor fragment chosen on the basis of previous structural studies represents a reliable model system to monitor the ability of CCK8 or CCK8 analogs to bind the cholecystokinin receptor. Together with previous studies, this confirms that the receptor fragment approach adopted to define the binding mode of the CCK8 fragment of cholecystokinin with its two receptors, CCK(A) and CCK(B,) can be used to characterize the binding from the equilibrium standpoint. In this context, fluorescence spectroscopy proves to be the favored technique to measure dissociation constants in the nanomolar to micromolar range.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles

“…Change in fluorescence emission of the enzyme was observed in relation to increasing concentration of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and STX. The reduction in intensity of fluorescence (Δ F ) at each concentration was normalized against the total maximum fluorescence of the enzyme ( F max ). , Data was directly fitted with a nonlinear least-squares fitting in the program PRISM ( ), with one ligand binding site with no interaction between sites since these provided the best fit.…”

“…To characterize the binding of a putative substrates of SxtN, the intrinsic fluorescent emission signature of tryptophan was measured between 300 and 500 nm at 22 °C when excited at 295 nm (5 mm slit), subtracting the fluorescence of the dialysis buffer as a blank (CARY eclipse fluorescence spectrophotometer, Varian). 29 Change in fluorescence emission of the enzyme was observed in relation to increasing concentration of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and STX. The reduction in intensity of fluorescence (ΔF) at each concentration was normalized against the total maximum fluorescence of the enzyme (F max ).…”

“…The reduction in intensity of fluorescence (ΔF) at each concentration was normalized against the total maximum fluorescence of the enzyme (F max ). 29,30 Data was directly fitted with a nonlinear least-squares fitting in the program PRISM (www. graphpad.com 31 ), with one ligand binding site with no interaction between sites since these provided the best fit.…”

Synthesis and Evaluation of a Fluorescent Non‐Peptidic Cholecystokinin‐B/Gastrin Receptor Specific Antagonist for Cancer Cell Imaging

“…The receptor fragment approach is also suitable for the characterization of binding between peptide (or nonpeptide) ligands and receptor fragments from the equilibrium standpoint 6. Thus, fluorescence spectroscopy has proved to be the favored technique to measure dissociation constants in the nanomolar to micromolar range 7…”

“…Fluorescent ligand spectroscopy has been used for the characterization of ligand‐binding domains and conformational changes that occur in multiple members of this superfamily, including rhodopsin, the β 2 adrenergic receptor, and the CCK receptor 6. 7 Both CCK A and B receptors (CCKARs and CCKBRs) have been shown to undergo ligand‐induced internalization in pancreatic acini23 and transfected NIH 3T3 cells,24 respectively. In a study using fluorescence to gain insight into differences between active and inactive conformations of the receptor, two series of fluorescent probes were developed, built on a structurally related peptide agonist and antagonist of the CCK receptor 25…”