Bioactive Sequences Research Articles

Cloning of a cDNA Encoding Porcine Brain Natriuretic Peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.

Integral membrane glycoprotein properties of the prohormone pro-transforming growth factor-alpha.

Transforming growth factor-alpha (TGF-alpha) is a mitogenically active polypeptide hormone produced by acutely proliferating neoplastic and embryonic tissues. Molecular cloning of TGF-alpha cDNA from human and rat has indicated that this factor is synthesized as part of a larger precursor, proTGF-alpha. An intriguing feature of proTGF-alpha is that it contains a sequence of 24 hydrophobic amino acids following the bioactive TGF-alpha sequence. Similar hydrophobic sequences present in transmembrane proteins are cotranslationally anchored into the phospholipid bilayer of the rough endoplasmic reticulum, suggesting the possibility that pro TGF-alpha might be an integral membrane protein. Other secretory proteins including all known prohormones lack membrane anchoring sequences and are completely translocated across the endoplasmic reticulum membrane. To address the question of whether proTGF-alpha is as an integral membrane protein we have translated rat proTGF-alpha mRNA transcripts in the presence of rough endoplasmic reticulum membrane vesicles. The results indicate that proTGF-alpha is synthesized as an integral membrane glycoprotein. The 50-amino-acid TGF-alpha sequence in proTGF-alpha is exposed to the fluid extracellular phase where it can be released after cleavage by the appropriate enzyme.

Localization of a peptide identified by antibodies to gastrin/CCK in the gut of Cancer magister

A gastric peptide from the Dungeness crab (Cancer magister), extracted and characterized previously (Larson and Vigna 1983b), was localized in the foregut (stomach) of this species by immunocytochemistry using antisera specific for the bioactive carboxy-terminal amino acid sequence common to gastrins and cholecystokinins (CCKs). Immunoreactivity was found in all gastric epithelial cells and in the procuticle. Electron microscopy revealed an absence of peptidergic secretory granules in the gastric epithelial cells. The pattern of immunostaining suggests that the gastric epithelial cells secrete this peptide apically where it is incorporated into the cuticle lining the lumen. Specific immunostaining could not be demonstrated in various neural ganglia or in the hypodermis. The distribution of this peptide is different from that of gastrin/CCK in vertebrates and other invertebrates. This suggests that the crab gastric peptide is sufficiently similar to gastrin/CCK to react with C-terminal specific antisera, but may be anatomically, functionally, and possibly phylogenetically otherwise unrelated.

Gastrin/cholecystokinin-like immunoreactive peptides in the Dungeness crab, Cancer magister (Dana): immunochemical and biological characterization

The purpose of this investigation was to characterize a gastrin/cholecystokinin-like immunoreactant (G/CCK-LI) extractable from the crab, Cancer magister. G/CCK-LI was extracted best in boiling water and was found mainly in the stomach, hemolymph and carapace. A relatively large immunoreactive peptide in the stomach and apparently smaller forms in the hemolymph and carapace were separated by Sephadex G-50 fractionation. Anion-exchange chromatography further fractionated the stomach form into three major peaks. The crab material cross-reacted with three antisera specific for the common C-terminus of gastrin/CCK, but cross-reacted much less with three antisera directed against other portions of the gastrin molecule. Partially purified crab stomach G/CCK-LI inhibited the binding of labeled CCK to mouse brain G/CCK receptors but not to rat pancreatic CCK receptors. The crab peptide did not stimulate rat gastric acid or rat pancreatic amylase secretion. These results indicate that the crab peptides are structurally similar to, but distinguishable from, the bioactive C-terminal amino acid sequence common to gastrins and CCKs.

Residue-specific radioimmunoanalysis: a novel analytical tool. Application to the C-terminus of CCK/gastrin peptides

Five antisera directed against the common bioactive C-terminal tetrapeptide sequence of cholecystokinin (CCK) and gastrin were examined with respect to the significance of each residue for the antibody binding. Systematic substitutions and/or derivatizations of each of the four residues showed a unique pattern for each antiserum although they were raised against the same antigen and have the same sequence-specificity. The pattern of reactivity towards the related cardioexcitatory FMRF amide peptide, and analogues hereof confirmed the residue specificity of the antisera. While it is well known that even small covalent modifications of the antigen can influence the antibody binding profoundly, the great variations in significance of each residue among randomly selected antisera raised against the same antigen and specific for the same sequence has not been known so far. Hence, by appropriate combination of antisera their different residue specificity can be used for detection of amino acid substitutions or modifications. Such immunochemical sequence analysis requires only femto- or picomolar amounts of peptides, which need not necessarily be purified. Thus, residue-specific immunoanalysis may be a versatile tool in studies of species differences, phylogenesis and synthesis of peptides.