Gj. Dockray

Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay.

Recent studies on the gene sequence encoding the human pyloric antral hormone, gastrin, indicate a precursor of 101 residues. We have now raised antibodies to a synthetic analogue corresponding to (Tyr)-human progastrin COOH-terminal pentapeptide. The antibodies could be used in radioimmunoassay to measure this peptide, but they did not react with corresponding fragments of procholecystokinin, porcine progastrin, or other human progastrin-derived peptides, notably heptadecapeptide gastrin (G17), and 34-residue gastrin (G34). Radioimmunoassay of human antral and duodenal extracts revealed a major peak of activity that corresponded to the native COOH-terminal fragment of progastrin, and occurred in approximately equimolar amounts with COOH-terminal G17 immunoreactivity. In addition, there was a minor peak of apparently higher molecular weight material. In some gastrinomas the latter material was the predominant immunoreactive form, and it occurred in higher molar concentrations than any other form of gastrin. Digestion of this material with trypsin liberated peptides that reacted with antibodies specific for the NH2-terminus of G34, and G17. On this basis the high molecular weight component was identified as a form of gastrin that extended from the COOH-terminus of the precursor to a point at least beyond the NH2-terminus of G34, and probably included the entire progastrin sequence. The results suggest differences in posttranslational processing pathways of progastrin in antrum, duodenum, and gastrinomas. They also indicate that the present experimental approach allows the identification of progastrin-like substances, which should open the way to studying the mechanisms of gastrin biosynthesis.

Comparison of the Metabolism of Sulfated and Unsulfated Heptadecapeptide Gastrin in Humans

The metabolism of synthetic human sulfated heptadecapeptide gastrin (G-17) was studied in normal human volunteers. Plasma concentrations were measured by radioimmunoassay using antibodies specific for intact G-17, and for the C- and N- terminus of G-17, during and after infusion of both sulfated and unsulfated G-17. With all three antibodies, plasma concentrations at a steady state were higher during infusion of sulfated compared with unsulfated G-17. In addition, the half-life in plasma measured by the three antibodies was two to five times higher for sulfated G-17 compared with unsulfated G-17. The half-life measured by N-terminal-specific antibodies was greater than that with antibodies specific for C-terminal or intact G-17. The difference was accounted for by the production during infusion of N-terminal fragments of relatively long half-life. The pattern of fragments generated during infusion of sulfated G-17 resembled that during unsulfated G-17 infusion, but there was no evidence of desulfation in the systemic circulation. The results indicate that in humans, sulfation protects G-17 from metabolism.

Isolation and Characterization of the Intact Gastrin Precursor From a Gastrinoma

Antibodies to the extreme C‐terminal region of human progastrin have been used to monitor the isolation of high‐M r immunoreactive material in a gastrinoma extract. Microsequence analysis of the product revealed amino acid residues in the first 18 positions corresponding to those predicted from the cDNA sequence for preprogastrin starting at position 22; the sequence and immunochemical data together allow the identification of this material as intact progastrin. Implications for gastrin biosynthesis are discussed.

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.

Isolation, structure and properties of the C-terminal flanking peptide of preprocholecystokinin from rat brain

The C‐terminal flanking peptide of preprocholecystokinin has been isolated from rat brain. Micro‐sequence analysis revealed the primary structure: Ser‐Ala‐Glu‐Asp‐Tyr‐Glu‐Tyr‐Pro‐Ser. Arylsulphatase and mild acid hydrolysis suggested that both tyrosine residues are sulphated. The peptide was not active in bioassay systems that respond to CCK8; the significance of the conserved tripeptide Ser‐Ala‐Glu is discussed.