Post-translational processing of cholecystokinin in pig brain and gut.

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A radioimmunoassay was developed to detect the cholecystokinin (CCK)-associated nonapeptide (CAP-9) that forms the COOH terminus of pig preproCCK. This peptide (Ser-Ala-Glu-Glu-Tyr-Glu-Tyr-Thr-Ser) is presumably produced at the time that the tyrosine-sulfated octapeptide CCK8(s) is cleaved from preproCCK. Radioimmunoassay of a dried methanol extract of pig brain revealed no detectable CAP-9 immunoreactivity, whereas acid desulfation of the dried methanol extract prior to radioimmunoassay resulted in easily measurable concentrations of CAP-9 immunoreactivity. Two peptides, CAP-9 and des-Ser9-CAP-9, were purified from a methanol extract of 8 kg of commercially obtained whole pig brains. Amino acid analysis showed that each peptide has both tyrosines sulfated. Thus, the likely sequence of CCK posttranslational processing events is sulfation ofthe three tyrosines in the COOH terminus of preproCCK followed by peptide cleavage and appearance of CCK8(s) and CAP-9(s,s).Cholecystokinin (CCK) is unique among the brain-gut peptides in that comparable concentrations of immunoreactive CCK are found in both anatomical locations (1-6). However, the distribution of molecular forms differs between brain and duodenum. Pig brain contains primarily the CCK COOHterminal octapeptide (CCK8) and the desoctapeptides of several larger forms: CCK33, CCK39, and CCK58 (7). In the duodenum the intact larger forms and desoctapeptides are about equally prominent (8). CCK8 in both brain and gut has an O-sulfated tyrosine in position 7 from the COOH terminus and a COOH-terminal phenylalanine amide.The cDNA sequences encoding preproCCK are now known for rat (9), pig (10), man (11), and mouse (12). All of them predict a Gly-Arg-Arg bridge to a nine amino acid peptide located on the COOH terminus of preproCCK. This CCK-associated nonapeptide (CAP-9), which in pig has the sequence Ser-Ala-Glu-Glu-Tyr-Glu-Tyr-Thr-Ser (10), should appear simultaneously with the formation of CCK8. It has recently been noted that O-sulfation of tyrosine residues in peptide sequences frequently occurs in tyrosines preceded by or in the vicinity of acidic amino acids (13). Since CAP-9 has two tyrosines, both of which are preceded by acidic amino acids, we hypothesized that one or both of these tyrosines might be sulfated. In this report we describe the purification, amino acid analysis, and sequence of the COOH-terminal nonapeptide of pig preproCCK and demonstrate that in pig brain the tyrosines of CAP-9 are fully sulfated. In addition we show that there is a fully sulfated octapeptide of the same sequence but lacking the COOH-terminal serine. MATERIALS AND METHODSRadioimmunoassay (RIA). CAP-9 was synthesized on a solid-phase support. The synthetic CAP-9 was dissolved in phosphate buffer and conjugated to thyroglobulin with glutaraldehyde at a ratio of 1.25 mg per 13 mg, respectively. The conjugate was purified on a small Sephadex G-25 column and was used to immunize each of two rabbits at monthly intervals with =100 ug of conjugated CAP-9. After the third immuniz...

Section: Discussionmentioning

confidence: 99%

“…Cholecystokinin (CCK) is unique among the brain-gut peptides in that comparable concentrations of immunoreactive CCK are found in both anatomical locations (1)(2)(3)(4)(5)(6). However, the distribution of molecular forms differs between brain and duodenum.…”

mentioning

confidence: 99%

Cholecystokinin-associated COOH-terminal peptides are fully sulfated in pig brain.

Eng¹,

Gubler²,

Raufman³

et al. 1986

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“…Cholecystokinin (CCK) is unique among the gut-brain peptides in that neuronal and mucosal tissues contain comparably high concentrations (1)(2)(3)(4)(5)(6). Nonetheless, the distribution among peptide forms differs between brain and gut.…”

mentioning

confidence: 99%

Pig brain contains cholecystokinin octapeptide and several cholecystokinin desoctapeptides.

Eng¹,

Shiina²,

Pan³

et al. 1983

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A sequential method employing methanol extraction of the COOH-terminal frgment of cholecystokini (CCK) from pig brain followed by HCI extraction of the more basic CCK peptides was used as the first step in purification of these peptides. Recovery was monitored with two different assays, one directed to the COOH terminus of CCK and the other to the NH2 terminus. The amino acid content and sequence were determined for each of five peptides after purification. The only peptide containing COOH-terminal immunoreactivity was CCK-octapeptide (CCK8). The other four peptides did not contain CCK8 and had lost one or two additional amino acids, perhaps as a consequence of the action of carboxypeptidases. These peptides were shown to be CCK33-desnonapeptide, CCK39-desnonapeptide and -desdecapeptide, and a large molecular weight precursor, CCK58-desnonapeptide, containing 19 amino acids (Ala-Val-Gln-Lys-Val-AspGly-Glu-Ser-Arg-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg) NH2-terminal to CCK39. The the NH2-terminal fragments of CCK58, CCK39, and CCK33 were about equally prominent. The brain, unlike the gut, appears to cleave CCK8 rapidly from a precursor peptide but to process the NH2-terminal portions of the molecule more slowly and incompletely.Cholecystokinin (CCK) is unique among the gut-brain peptides in that neuronal and mucosal tissues contain comparably high concentrations (1-6). Nonetheless, the distribution among peptide forms differs between brain and gut. In an earlier report of a sequential extraction method using methanol to extract COOH-terminal fragments of CCK followed by HCI to extract intact CCK and its NH2-terminal fragments, we failed to find significant amounts of a CCK peptide with both COOHand NH2-terminal immunoreactivity in brain extracts although such peptides, presumably CCK33, CCK39, or both and a larger precursor peptide were prominent in gut extracts (6). In this report we describe the purification and sequences of several CCK peptides from pig brain and again observe the absence of detectable amounts of intact CCK in brain. Preliminary experiments showed that 0.02 M HCI had over 80% of the efficiency of 0.1 M HCI in extracting immunoreactive CCK from the methanol filter cakes. However, 0.02 M HCI extracted less than 10%o as much protein and therefore was used as the acid extractant. Ten methanol filter cakes were each extracted with 450 ml of 0.02 M HCl in a Waring blender. Each extract was centrifuged to remove the precipitate and the supernatants were pooled prior to further purification. MATERIALS AND METHODSPurification. Methanol extracts. Chromatographic runs and recoveries were monitored with the R71 RIA because only COOH-terminal immunoreactivity was extracted by methanol. The methanol extract (5 liters) was pumped through a 230-ml column of DEAE-Sepharose (Pharmacia) at 60 ml/hr. The column was washed with distilled water and elution was effected with a gradient from water to 1 M ammonium acetate (pH 5.5) in a total volume of 3 liters. Peak fractions of immunoreactivity (400 ml) were...

“…Pig and dog intestinal mucosa CCK forms differ from brain forms by the presence of significant amounts of intact CCK-33 and -39. Brain CCK may be more quickly processed from large (CCK-58) to intermediate (CCK-39 and -33) to small forms (CCK-8 and -5), or there may be another set of enzymes present that directly converts the brain large forms into the small forms (21). The major CCK-LI peak that is eluted near the salt region on Sephadex G-25 gel permeation columns is the carboxyl-terminal octapeptide of CCK; the minor, diffuse peak that is eluted after CCK-8 is presumably the carboxyl-terminal pentapeptide of CCK.…”

Section: Discussionmentioning

confidence: 99%

Isolation of a large cholecystokinin precursor from canine brain.

Eysselein¹,

Reeve²,

Shively³

et al. 1984

Cholecystokinin (CCK)-like immunoreactivity (CCK-LI) in a pool of 12 dog brains was extracted sequentially into boiling water and cold 2% trifluoroacetic acid. Gel filtration on Sephadex G-50 revealed three main molecular forms detected by a carboxyl-terminal antibody; one was eluted in the position of CCK-58 (58 amino acid residues long); a second, in the position of CCK-8; and a third, near the radioactive iodide marker. When the CCK-LI was purified by affinity chromatography using carboxyl-terminal CCK antibody followed by three steps of reversed-phase high-pressure liquid chromatography, three components were isolated and characterized by sequence microanalysis. The smallest component was the pentapeptide common to gastrin and CCK. The second peak was eluted in the same region as synthetic CCK octapeptide, and sequence analysis showed that the chemical structure of this biologically active region of canine CCK is identical to that found in sheep and pig brains. The 22-residue aminoterminal sequence of brain CCK-58 was: Ala-Val-Gln-LysVal-Asp-Gly-Glu-Pro-Arg-Ala-His-Leu-Gly-Ala-LeuLeu-Ala-Arg-Tyr-Ile-Gln-, the same as the sequence found for canine intestinal CCK-58 from this pool of dogs. This is the same sequence others have reported for porcine brain CCK-58 lacking nine amino acid residues (CCK-58 desnonapeptide) except that the porcine peptide had a serine in position 9. The canine CCK amino-terminal sequence differed from the se-