Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin‐58 and their action on gallbladder contraction

Abstract

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...

Unique activities of cholecystokinin-58; physiological and pathological relevance

“…Other unique roles for CCK58 compared with shorter forms (CCK8 or CCK33) have previously been demonstrated in the modulation of central catecholaminergic mechanisms and neuroendocrine functions [33], the pattern of gallbladder muscle contraction [34,35], and in intestinal afferent nerve discharge [36]. More recently, it was shown that CCK58 inhibited food intake much more strongly than CCK8 by profoundly increasing the intermeal interval (S.W.…”

Nonsulfated cholecystokinins in the small intestine of pigs and rats

“…First, canine thyroid C-cells contain mainly nonsulfated CCK-8 and -5 [6], and CCK in human medullary thyroid carcinomas is all nonsulfated CCK-8 [7]. Later it was shown that CCK-58 in the porcine and canine small intestine occurs in both sulfated and nonsulfated form [8,9]. Thus, nonsulfated CCK peptides seem to occur in vivo.…”

Topical Cholecystokinin Depresses Itch-Associated Scratching Behavior in Mice

“…In various CCKs, sulfation of the tyrosine at position 7 from the C-terminus is a posttranslational modification that makes them biologically active peptides (Ma et al, 2006). Varying lengths of CCKs with or without sulfate have been studied for their activities as gastrointestinal hormones (Bonetto et al, 1999) and as CNS players (Rehfeld et al, 2007). Two types of CCK receptors have been identified: CCK1R (CCK-A receptor) and CCK2R (CCK-B receptor).…”