Purification, amino acid sequence, synthesis, and receptor selectivity of alligator gastrin

Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18 Sep-Paks, Sephadex G-50, pH stable C8 reversed-phase HPLC, and C18 reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin.