Preparation of an N-acetyl-octapeptide of cholecystokinin. The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues.
The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5--4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentration-dependent isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.
D000107:Acetylation; D000818:Animals; D002415:Cats; D002766:Cholecystokinin / Q000302:isolation & purification / Q000378:metabolism*; D005704:Gallbladder / Q000378:metabolism; D006358:Hot Temperature; D007525:Isoelectric Focusing; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D012844:Sincalide
D010446:Peptide Fragments; D002766:Cholecystokinin; D012844:Sincalide