Identification of gastrin component I as gastrin‐71

Abstract

Gastrin component I is the largest hormonally active form of gastrin. In order to determine its structure, we isolated progastrin-derived peptides from normal human antral tissue. A radioimmunoassay specific for sequence 20-25 of human progastrin was developed to monitor the purifications. After four or five steps of reverse-phase chromatography, the peptides were pure and could be identified by a combination of microsequence, amino acid and mass spectral analysis as well as by a library of sequence-specific immunoassays. In addition to intact progastrin 1-80, fragments 1-71, 1-35, 6-35, 20-35, and 20-36 of progastrin were identified. Only the 71-amino-acid peptide contained at its C-terminus the a-amidated bioactive site (Trp-Met-Asp-Phe-NH,). This unoheptacontapeptide amide (gastrin-71) corresponds to component I and is the largest possible bioactive product of progastrin. Its structure shows that progastrin is used in its entirety for biosynthesis of active peptides. The occurrence of fragments 6-35,20-35, and 20-36 demonstrate that antral progastrin is partially cleaved at two monobasic sites (Arg5 and Argl9) in addition to processing at the three C-terminal dibasic sites. The results show that both the N-and C-terminal parts of antral progastrin undergo extensive processing. The results also suggest that progastrin may follow two different processing pathways of which the less trafficked releases gastrin-71.Biologically active peptides (hormones, neuropeptides, cytokines, and growth factors) are characterized by high specific bioactivity at low concentrations. The peptides are synthesized from long precursors, and achieve their unique bioactivity only after elaborate and precise precursor processing. During the last decade much information about tissue-specific precursor processing has accumulated (for review, see

Gastrin, cholecystokinin and gastrointestinal tract functions in mammals

“…Other small COOH-terminal fragments of fourteen (G14 or mini gastrin) and six residues (G6) may occur in tissues (Gregory et al 1979(Gregory et al , 1983), but are not thought to be functionally important. In man, relatively small amounts of the NH 2 -terminally extended form of G34 may occur which is now known to correspond to a seventyone-residue peptide (Rehfeld & Johnsen, 1994). A large form of gastrin originally named 'big big gastrin' is now thought to be an artifact.…”

Biologically Active Recombinant Human Progastrin6–80Contains a Tightly Bound Calcium Ion

“…Small amounts of progastrin (less than 1 nmol/gm tissue) have been isolated from human tissues (10), but the largest progastrin-derived peptide available in bulk to date via organic synthesis is progastrin 20 -71 (10). The related prohormone procholecystokinin (pro-CCK) 1 has been expressed with an N-terminal histidine tag and purified from baculovirusinfected insect cells (11).…”

“…With the exception of the well known zinc-dependent polymerization of insulin and proinsulin (15), this is the first report of selective, high affinity binding of a metal ion to a prohormone. (17) and hence encoding the entire sequence of mature human progastrin (10), was subcloned into HindIII-cut and dephosphorylated pGEX-2TH (18). Clones with the insert in the correct orientation were selected by restriction mapping.…”

Metabolism of recombinant progastrin in sheep

“…The antiserum detects the pentadecapeptide 20-33 and progastrin-(20-72) amide equally well. Progastrin fragments extended NH 2 terminally to 20-33 have ϳ50% cross reactivity, whereas cross reactivity to progastrin fragments without amino acid 20 is 10% and without residues 20 and 21 is 0.1% (30). Antiserum 88235 (diluted to 1:500,000) was used to measure progastrin in Sephadex G 50 column fractions against a buffer A standard curve constructed with Tyr 34 -progastrin-(20-34) (kindly supplied by Jens Rehfeld) as standard and [ 125 I]Tyr 34 -progastrin-(20-34) as tracer.…”

“…We are grateful to Prof. Jens Rehfeld (Rigshopitalet, Copenhagen, Denmark) for kindly donating antisera 8017, 7270, and 88235 and human progastrin- (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). …”