Natural and synthetic CCK-58. Novel reagents for studying cholecystokinin physiology

CCK-58 is a unique reagent for testing how segments of a peptide far removed from its active site can influence the expression of its biological activity. Indications of tertiary structure have come from studies with natural peptide purified from canine small intestine. These studies gave clear indications that tertiary structure affects CCK-58 bioactivity, but the small quantities of CCK-58 available made it impossible to characterize completely how tertiary structure influenced bioactivity. Canine CCK-58 was synthesized manually using a solid support and was purified by reverse phase high pressure liquid chromatography (HPLC). Synthetic CCK-58 was characterized by isocratic reverse phase and gradient HPLC, amino acid analysis, mass spectral analysis, sequence analysis, and three bioassays. Synthetic and natural canine CCK-58 had the same elution profiles, amino acid composition, sequence, and mass. The two peptides were equipotent for the stimulation of pancreatic secretion. Natural canine CCK-58 was equipotent to CCK-8 for CCK "B" receptor binding, a further indication of the purity of the natural peptide. However, natural CCK-58 was more potent than CCK-8 for CCK "A" receptor binding and less potent than CCK-8 for stimulation of pancreatic secretion. These data support the concept that CCK-58 has a stable tertiary structure. This structure does not affect its binding to CCK "B" receptors, enhances its binding to low affinity CCK "A" receptors, and decreases its activity expressed through binding to high affinity CCK "A" receptors. The concept of a stable tertiary structure is also supported by the fact that many antibodies directed towards the carboxyl terminus of cholecystokinin react better with CCK-8 than CCK-58. The availability of synthetic CCK-58 will allow analysis of its tertiary structure by physical and chemical methods as well as studies on how peptide tertiary structure can affect receptor binding, receptor activation, metabolism in blood, degradation in interstitial fluid, and inactivation at the receptor. Evaluating all of these systems will help investigators understand the regulation of cholecystokinin activity by its major endocrine form, CCK-58.