The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.     

Isolation and characterization of bovine pancreastatin

Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248-294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.     

Peptide YY. Structure of the precursor and expression in exocrine pancreas

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.     

PHI--a new brain-gut peptide

The detection of the C-terminal amide structure in porcine intestinal extracts has led to the discovery of a 27 amino acid residue peptide designated PHI (PHI-27, peptide HI). The peptide was found to have structural homologies to vasoactive intestinal peptide (VIP) and growth hormone-releasing factor (GRF). Subsequent studies have revealed that PHI exhibits a variety of biological activities which resemble those of VIP. Moreover, it was found that the peptide is able to inhibit the binding of VIP to its receptors, and to stimulate cyclic AMP production. PHI is present in both brain and gut in high concentrations and probably acts as a neurotransmitter or neuromodulator rather than a hormone. A comparison of the amino acid sequences of porcine, human and bovine PHI indicated that human PHI differs from the porcine peptide in two positions (12 and 27), and bovine PHI differs in one position (10). The amino acid sequence (deduced from the cDNA sequence) of the VIP precursor recently obtained from human neuroblastoma cells also contains an identical sequence to the newly-isolated human PHI from human colonic extracts. PHI has thus been shown to be co-synthesized with VIP in the same precursor molecule.     

Characterization of a peptide released during the reaction of human alpha 1-antitrypsin and bovine alpha-chymotrypsin.

The release of a peptide (molecular weight: about 3,600) was observed during complex formation between human alpha 1-antitrypsin (alpha 1-AT) and bovine alpha-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular weight were also released during the complex formation of alpha 1-AT with bovine trypsin or porcine elastase. The peptide released from the complex with bovine alpha-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine alpha-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original alpha-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native alpha 1-AT. These results suggest that the peptide was released from the C-terminal region of alpha 1-AT uon interaction with alpha-chymotrypsin. It is tempting to suggest that alpha 1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.     

Purification and characterization of chymodenin. A hormone-like peptide from porcine duodenum

Chymodenin, a hormone-like duodenal peptide which rapidly alters the proportions of secreted pancreatic digestive enzymes to a mixture relatively richer in chymotrypsinogen than that found in basal secretion, has been purified to homogeneity. The starting material was an acidic methanol-soluble, neutral pH-insoluble fraction of an acetic acid extract of porcine duodenum; the purification consisted of cation-exchange chromatography on SP-Sephadex and CM-Sephadex in ammonium bicarbonate gradients, and gel filtration on Sephadex G-75 in dilute acetic acid. The yield of material was followed by radioimmunoassay. Homogeneity was determined from chymodenin's behavior in disc gel electrophoresis in an acidic counter-migration-of-dye system, sodium dodecyl sulfate-urea gels, gel filtration, dansyl-Edman reaction, reversed-phase high pressure liquid chromatography, isotachophoresis, and sedimentation equilibrium ultracentrifugation. The electrophoretic mobility, the molecular weight of 9,000-10,000, and the biological activity differed from those of other gastrointestinal peptide hormones. The amino acid composition was unique. Chymodenin is the first purified hormone-like substance reported capable of altering the composition of the mixture of secreted digestive enzymes, independent of the stimulation of massive pancreatic protein output.     

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.     

Isolation from porcine intestinal extracts of a cholecystokinin-like peptide and a peptide with homology to cytochrome oxidase polypeptide VII and chymodenin

A number of different forms of cholecystokinin (CCK) exist in the brain and intestine. Gel permeation and ion exchange chromatography and high performance liquid chromatography have been used to isolate a peptide from partially purified porcine intestinal extracts with N-terminal homology to porcine brain CCK-58. This peptide contracted both the guinea pig ileum longitudinal muscle and gallbladder muscle and these responses were inhibited by dibutyryl cyclic GMP or proglumide. The potency was approximately 1/100 of that of CCK-8. The reason for this low potency is unclear, but it is possible that a critical part of the biologically active region is modified or that it is a truncated form of CCK-58. A further peptide was isolated with a sequence homologous to cytochrome oxidase polypeptide VII and chymodenin.     

Somatostatin action on insulin secretion induced by chicken and porcine vasoactive intestinal peptide in the perfused rat pancreas

The isolated perfused rat pancreas with duodenal exclusion was used to study the stimulation of glucose-induced insulin release in response to chicken and porcine vasoactive intestinal peptide (VIP). The insulin response to 5.5 or 16.7 mM glucose was markedly enhanced by 750 pM porcine VIP and a concentration of 250 pM was still effective. At 250 pM, chicken VIp exhibited a slightly higher potency than porcine VIP at both glucose concentrations. The main difference between the two peptides was that the effect of porcine VIP disappeared immediately after the peptide suppression but tha of chicken VIP persisted for an additional period of 8-10 min. Somatostatin (10 ng/ml) blocked the stimulatory effect of both VIP molecules on glucose-induced insulin secretion. After suppression of VIP and somatostatin from the perfusion medium, insulin release increased to levels higher than those with glucose alone in the case of the avian peptide, but not in that porcine VIP. The data are consistent with previous results in the literature on stimulation of exocrine pancreas secretion and interaction with intestinal epithelium.     

Actions of a new peptide from porcine intestine (PHI) on pancreatic secretion in the rat and turkey

A newly purified peptide from porcine intestine (PHI) was found to be a potent stimulant of the flow of pancreatic juice in anaesthetized turkeys (D₅₀ 75 ng/kg), and was about half as active as chicken vasoactive intestinal peptide (VIP, D₅₀ 47 ng/kg) to which it is structurally related. However, in anaesthetized rats, PHI, like VIP, was a weak stimulant of the flow of pancreatic juice. In contrast, porcine secretin, which is also structurally related to PHI, is a potent stimulant of the rat pancreas and a weak stimulant in birds. PHI had less than 0.01% immunochemical potency in a VIP radioimmunoassay. We conclude that PHI has VIP-like rather than secretin-like actions on the pancreas in birds and mammals; an avian counterpart of PHI could play a physiological role in the control of the exocrine pancreas.     

The primary structure of peptide Y (PY) of the spiny dogfish, Squalus acanthias: immunocytochemical localisation and isolation from the pancreas.

1. Endocrine cells within islets, exocrine parenchyma and ductal epithelium in the pancreas of the spiny dogfish, Squalus acanthias, were immunostained with an antiserum to the C-terminal region of mammalian neuropeptide Y (NPY). 2. Radioimmunoassay of pancreatic extracts with the same antiserum detected immunoreactivity in the dorsal lobe (338 pmol/g) and ventral lobe (433 pmol/g). Reverse phase HPLC analysis of both extracts resolved a single immunoreactive peptide. 3. The primary structure of the isolated peptide was established as: YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY.NH2. 4. Peptide Y (PY) from Squalus acanthias is identical in primary structure to an NPY-related peptide isolated from the pancreas of Scyliorhinus canicula and has a 31/36 residue homology with porcine NPY. The 5 substitutions are highly-conservative.     

Investigation of the requirements for O-glycosylation by bovine submaxillary gland UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosamine transferase using synthetic peptide substrates

Peptides containing a triprolyl sequence carboxyl to a threonine residue can be O-glycosylated by a crude Triton x-100 extract of porcine submaxillary glands (Young, J. D., Tsuchiya, D., Sandlin, D. E., and Holroyde, M. J. (1979) Biochemistry 18, 4444-4448). In the present paper, we have studied the characteristics of the O-glycosylating enzyme, UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosamine transferase, from a membrane extract of bovine submaxillary glands using 11 synthetic peptide substrates in which the Thr-Pro-Pro-Pro was varied. The effect of these changes was measured by determining the apparent Km and Vmax values of the substrates. The studies thus far reveal: threonine cannot be glycosylated without a carboxyl triprolyl sequence; the alpha amino acid group of the threonine must be blocked; the nature of the group NH2-terminal to the threonine affects the kinetics of the reaction; and one residue can be between the threonyl and the triprolyl sequence. The triprolyl sequence in a protein may be an important signal for O-glycosylation.     

Purification of bovine cholecystokinin-58 and sequencing of its N-terminus

Molecular cloning of cholecystokinin (CCK) mRNA from porcine brain and gut has demonstrated that CCK is synthesized as an identical precursor in both tissues. The sequence for porcine CCK-58 predicted from CCK cDNA was identical with the amino acid sequence of the peptide purified from different lots of animals. However one group did report that there were differences in the N-terminus of CCK-58 purified from the intestines of two different lots of mongrel dogs. In the current report it is demonstrated that the amino acid sequences of CCK-58 purified separately from three bovine brains are identical through the first 19 N-terminal amino acid residues. The peptides were sequenced for ten additional steps and were shown to be identical with the previously reported sequences for the N-terminus of CCK-39. The N-terminus of bovine CCK-58 has the following sequence: AVPRVDDEPRAQLGALLAR.     

The mechanism of activation of rabbit plasminogen by urokinase. Lack of a preactivation peptide

We have obtained direct evidence which we interpret to prove that an amino terminal peptide need not be released from rabbit plasminogen prior to its conversion to plasmin by urokinase. The single chain plasminogen molecule possesses an amino terminal amino acid sequence of NH₂-glu-pro-leu-asp-asp. When this plasminogen is activated to plasmin by urokinase in the presence of the Kunitz bovine trypsin-plasmin-kallikrein inhibitor (BTI), a two chain disulfide linked molecule of plasmin is obtained. The heavy chain of this plasmin is directly derived from the original amino terminus of plasminogen since it possesses the identical amino terminal sequence as does native plasminogen. When the same plasminogen activation is carried out in the absence of BTI, the heavy chain of the plasmin obtained has a molecular weight of 6,000–8,000 less than the heavy chain of the plasmin obtained in the presence of this inhibitor. In addition, the heavy chain of this latter plasmin has an amino terminal sequence which differs from the original native plasminogen. These data show, in agreement with others, that the activation of plasminogen by urokinase is accompanied by the loss of an amino terminal peptide from plasminogen but also show, in contrast to the human plasminogen system, that cleavage of the internal peptide bond, leading to plasmin formation, can occur without cleavage of the amino terminal peptide.     

Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex

A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.     

Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA

Chromogranin A (CGA), also referred to as secretory protein I, is an acidic protein that has been detected in all neuroendocrine cell types examined and is often present in large amounts relative to other secreted proteins. For example, CGA comprises at least 40% of the soluble protein of the adrenal chromaffin granule, and it appears to be the major secretory protein in the parathyroid secretory granules. CGA complementary DNAs (cDNAs) from bovine adrenal and pituitary have recently been cloned and sequenced and found to be nearly identical. A region of bovine CGA has a high degree of amino acid sequence identity to pancreastatin, a recently isolated porcine peptide that inhibits glucose-induced insulin secretion. This suggests that CGA may be a prohormone. We have cloned and sequenced a human cDNA encoding CGA. This human CGA cDNA has an overall 86% nucleic acid identity to the bovine cDNA. Like the bovine CGA cDNA, the human cDNA has little homology to pancreastatin at the 5' region of this peptide but significant amino acid homology to the carboxyl-terminal portion of pancreastatin where the biologic activity resides. There is an area within the pancreastatin region of human CGA and porcine pancreastatin with a 70% amino acid identity to the calcium-binding moiety of the E-F hand proteins such as parvalbumin and oncomodulin. These data suggest that CGA and pancreastatin may both be members of a larger family of calcium-binding proteins.     

Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin

A cDNA encoding a human endothelium-derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.     

Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y

The amino acid sequence of a peptide isolated from the Pacific salmon (Oncorhynchus kisutch) endocrine pancreas has been determined. This simple 36 residue peptide is a member of the pancreatic polypeptide family. It contains a C-terminal tyrosinamide and is more homologous with porcine neuropeptide Y (NPY) (83%) and peptide YY (75%) than any of the previously characterized pancreatic polypeptides (PP). This peptide appears to be the major but not the only representative of this family of peptides present in the endocrine pancreas of this fish. This peptide is referred to as salmon pancreatic polypeptide (salmon PP).     

The N-terminal portion of an erythrotropin-like peptide from fetal bovine serum has sequence homology with the LDL-receptor and two proteins of the Epstein-Barr virus

An erythrotropin-like peptide (ELP) and bovine serum erythrotropin (ET) coeluted after more than four chromatographic steps. ELP was separated from ET on gel permeation high pressure liquid chromatography due to its large molecular weight. The effect of ELP on the stimulation of [3H] Thymidine incorporation in cell cultures of fetal bovine liver was much lower than the effect of ET. The N-terminal amino acid sequence of ELP indicated that it has a group of amino acids identical to portions of the LDL-receptor and the hypothetical proteins BHLF1 and BOLF1 deducted from the DNA sequence of the Epstein-Barr virus. These results are consistent with the idea that cell surface proteins have structural homologies with secreted proteins or some growth factor-related peptides.