Isolation and characterization of biologically active and inactive cholecystokinin-octapeptides from human brain.
Cholecystokinin-like immunoreactivity (CCK-LI) in 0.9 kg human brain was extracted by 2% trifluoroacetic acid at 4 degrees C. Sephadex G50 gel filtration of crude extract revealed one main molecular form of CCK, detected by a carboxy-terminal antibody (5135), that eluted in the position of CCK8. When the CCK-LI in the extract was purified by affinity chromatography using another carboxyl-terminal CCK antibody followed by several steps of reverse phase high pressure liquid chromatography (HPLC), a component was isolated that was found by sequence analysis to be identical to the carboxyl-terminal CCK-octapeptide of porcine CCK33, isolated from intestinal mucosa, and to CCK-octapeptide, isolated from sheep brain. This component possessed comparable biological potencies to synthetic sulfated CCK8 in eliciting amylase release and in competitively displacing radioiodinated CCK33 from isolated mouse pancreatic acini. Furthermore, it exhibited a similar binding characteristic to CCK8 in binding to specific receptors on mouse brain cortical particulate preparations. On high pressure liquid chromatography another minor, earlier eluting immunoreactive peak was observed, which had the same amino acid composition and sequence as CCK8. These findings suggested that this material was oxidized CCK8. This earlier eluting component, exhibiting CCK8-like immunoreactivity, did not induce amylase release from acini and had no or minimal effect in inhibiting tracer CCK33 binding to receptors on isolated acini or on mouse brain cortical particulate preparations at the concentrations tested.
D000596:Amino Acids / Q000032:analysis; D000681:Amylases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D051379:Mice; D010179:Pancreas / Q000201:enzymology; D011869:Radioligand Assay; D012844:Sincalide / Q000302:isolation & purification*
D000596:Amino Acids; D000681:Amylases; D012844:Sincalide