Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.Keywords: cholecystokinin-58; gallbladder contraction assay; MALDI mass spectrometry.CCK was first isolated from porcine intestine as a 33-residue peptide, CCK-33, the C-terminal octapeptide of which, CCK-8, was responsible for the bioactivity [1]. In addition to CCK-33 and CCK-8, other forms with different lengths have been isolated from intestine, including CCK-39 [2], and from brain of several species [3,4]. CCK-58 represents the full-length bioactive form processed N-terminally by the cleavage of the signal and flanking peptides, and C-terminally by cleavage of the C-terminal flanking peptide. The C-terminus is amidated and a tyrosine residue near the C-terminus is usually sulphated. CCK-58 has been isolated from dog intestine [5] and subsequently from the intestines or brains of several other mammals [6,7], but not from pig intestine, despite several attempts. The nonsulphated form of CCK-58 has not been isolated previously and its bioactivities have not been determined.In dog and human, CCK-58 has been found to be the major molecular form stored in intestine and brain and also the major endocrine form [5,8±10]. Studies of CCK have largely been limited to the short forms, mainly CCK-8 and CCK-33, produced by chemical synthesis. CCK-58 has been shown to have structural and functional properties different from shorter forms [11,12]. However, production of CCK-58 by chemical synthesis presents technical problems because of its size and the sulphated tyrosine residue. The latter structural feature and presence of the C-terminal a-amide also complicate production of the polypeptide by recombinant methodology. Therefore, intestinal or brain tissues remain valuable sources for isolation of CCK-58.We now describe isolation of sulphated and nonsulphated forms o...

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PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60 may play a role in the central nervous system disorders involving dopamine dysregulation.

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