From batch to continuous chromatographic purification of a therapeutic peptide through multicolumn countercurrent solvent gradient purification.

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated. Moreover, the purification process can be completely automatized. Aim of this work is to present a simple procedure for the development of the MCSGP process based on a single batch experiment, in the case of a therapeutic peptide of industrial relevance. This allowed to recover roughly 90% of the injected glucagon in a purified pool with a purity of about 90%. A comparison between the performance of the MCSGP process and the classical single column batch process indicates that percentage increase in the recovery of target product is +23% when transferring the method from batch conditions to MCSGP, with an unchanged purity of around 89%. This improvement comes at the expenses of a reduction of about 38% in productivity.

A new strategy for recovery of two peptides without Glu employing glutamate-specific endopeptidase from Bacillus licheniformis.

The difficulty in the purification of bioactive peptide limited its application in food, drug and cosmetic industry. Here we report a new strategy for the recovery of two peptides employing glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL), which shows strong specificity for Glu residue. Human glucagon and human beta-defensin-2 (HBD-2) were peptides without Glu residue, and Glu residue was introduced between affinity tag and target peptide as recognition site of GSE-BL. Tagless human glucagon with the same HPLC retention time as native human glucagon and mature HBD-2 with antibacterial activity and cytotoxicity were obtained after GSE-BL treatment. This strategy has great potential in the recovery of bioactive peptide without Glu residue, thus facilitating large scale preparation of peptide and widening the application of bioactive peptide.

Influence of pressure and temperature on molar volume and retention properties of peptides in ultra-high pressure liquid chromatography.

In this study, pressure induced changes in retention were measured for model peptides possessing molecular weights between ∼1 and ∼4kDa. The goal of the present work was to evaluate if such changes were only attributed to the variation of molar volume and if they could be estimated prior to the experiments, using theoretical models. Restrictor tubing was employed to generate pressures up to 1000bar and experiments were conducted for mobile phase temperatures comprised between 30 and 80°C. As expected, the retention increases significantly with pressure, up to 200% for glucagon at around 1000bar compared to ∼100bar. The obtained data were fitted with a theoretical model and the determination coefficients were excellent (r(2)>0.9992) for the peptides at various temperatures. On the other hand, the pressure induced change in retention was found to be temperature dependent and was more pronounced at 30°C vs. 60 or 80°C. Finally, using the proposed model, it was possible to easily estimate the pressure induced increase in retention for any peptide and mobile phase temperature. This allows to easily estimating the expected change in retention, when increasing the column length under UHPLC conditions.

Expression, purification and preliminary characterization of glucagon receptor extracellular domain.

Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), one of class B G-protein coupled receptors (GPCRs). Diabetes is a bihormonal disease in which excessive glucagon secretion is a major contributor in the pathogenesis of this disease; elucidation of how glucagon binds to GCGR will facilitate the rational design of the GCGR antagonist for treating diabetic hyperglycemia. Here we report the successful expression and purification of the GCGR extracellular domain (GCGR-ECD) and its fusion protein with the glucagon peptide at its C-terminus (GCGR-ECD-Gc). We utilized the maltose binding protein (MBP) fusion method and disulfide bond isomerase DsbC co-expression approach for the success of the soluble expression of both GCGR-ECD and GCGR-ECD-Gc in Escherichia coli. We also obtained a high yield production of secreted GCGR-ECD with the baculovirus expression system by optimizing its N-terminal secreting signal. We first utilized isothermal titration calorimetry approach to determine the in vitro binding affinities of glucagon to the GCGR-ECD. No significant differences were found between the prokaryotic expressed GCGR-ECD (7.6µM) and the eukaryotic glycosylated one (6.6µM). The observation of the intra ligand-receptor binding within the fusion protein GCGR-ECD-Gc suggests it as a good candidate for further structural study.

Expression, purification, and PC1-mediated processing of human proglucagon, glicentin, and major proglucagon fragment.

To examine the cleavage specificity of different members of the furin/propeptide convertase (PC) family of enzymes, we have selected proglucagon (PG) as a model substrate. PG was selected because it is subject to differential processing in vivo. PG is thought to be cleaved initially at an interdomain site to produce glicentin and the major proglucagon fragment (MPGF). These intermediates are subsequently cleaved, most likely by the convertases PC2 and PC1, respectively. To determine the exact sites within PG that are cleaved by PC1 and PC2, we attempted to produce milligram quantities of human PG, glicentin, and MPGF for use in an in vitro conversion assay. A methionine residue was added to the N-terminus of each protein to initiate translation. Purification was achieved using cation exchange and reversed-phase chromatography, and the integrity of the methionylated proteins was confirmed by both electrospray ionization-mass spectrometry and amino acid analysis. The combined expression and purification scheme is fast, efficient, and results in milligram quantities of > or =95% pure proglucagon, > or =95% pure MPGF, and > or =93% pure glicentin. These prohormones are cleaved by PC1 to produce product peptides consistent with the processing of PG observed in vivo, and should therefore be suitable for further analysis of the post-translational processing of PG.

Establishment of clonal colony-forming assay system for pancreatic stem/progenitor cells.

Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as alpha and beta cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.

The Caenorhabditis elegans polarity gene ooc-5 encodes a Torsin-related protein of the AAA ATPase superfamily.

The PAR proteins are required for polarity and asymmetric localization of cell fate determinants in C. elegans embryos. In addition, several of the PAR proteins are conserved and localized asymmetrically in polarized cells in Drosophila, Xenopus and mammals. We have previously shown that ooc-5 and ooc-3 mutations result in defects in spindle orientation and polarity in early C. elegans embryos. In particular, mutations in these genes affect the re-establishment of PAR protein asymmetry in the P(1) cell of two-cell embryos. We now report that ooc-5 encodes a putative ATPase of the Clp/Hsp100 and AAA superfamilies of proteins, with highest sequence similarity to Torsin proteins; the gene for human Torsin A is mutated in individuals with early-onset torsion dystonia, a neuromuscular disease. Although Clp/Hsp100 and AAA family proteins have roles in diverse cellular activities, many are involved in the assembly or disassembly of proteins or protein complexes; thus, OOC-5 may function as a chaperone. OOC-5 protein co-localizes with a marker of the endoplasmic reticulum in all blastomeres of the early C. elegans embryo, in a pattern indistinguishable from that of OOC-3 protein. Furthermore, OOC-5 localization depends on the normal function of the ooc-3 gene. These results suggest that OOC-3 and OOC-5 function in the secretion of proteins required for the localization of PAR proteins in the P(1) cell, and may have implications for the study of torsion dystonia.

Islet hormones from the African bullfrog Pyxicephalus adspersus (Anura:Ranidae): structural characterization and phylogenetic implications.

The African bullfrog Pyxicephalus adspersus is generally classified along with frogs of the genus Rana in the subfamily Raninae of the family Ranidae but precise phylogenetic relationships between species are unclear. Pancreatic polypeptide (PP), insulin, and glucagon-like peptide (GLP-1) were isolated from an extract of P. adspersus pancreas and characterized structurally. A comparison of the amino acid sequence of Pyxicephalus PP (APSEPQHPGG(10)QATPEQLAQY(20)YSDLYQYITF(30)ITRPRF++ +. NH(2)) with those of the known amphibian PP molecules in a maximum parsimony analysis generates a single phylogenetic tree in which Pyxicephalus is the sister to the clade comprising the members of the genus Rana. The three orders of living amphibians form discrete clades with the representative of the Gymnophiona appearing as sister to the Caudata-Anura. In contrast, Pyxicephalus insulin (A chain, GIVEQCCHSA(10)CSLYDLENYC(20)N; B-chain, LANQHLCGSH(10)LVEALYMVCG(20)ERGFFYYPKS(30)) and and GLP-1 (HAEGTFTSDM(10)TSYLEEKAAK(20)EFVDWLIKGR(30)PK) resemble more closely the corresponding peptides from the cane toad Bufo marinus than the peptides from any species of Rana. Cladistic analysis based upon the amino acid sequences of insulin produced a polyphyletic assemblage with the Gymnophiona nesting within an unresolved clade containing the non-ranid frogs. The data support the assertion that the amino acid sequence of PP, but not those of the other islet hormones, is of value as a molecular marker for inferring phylogenetic relationships between early tetrapod species.

Purification and characterization of insulin and peptides derived from proglucagon and prosomatostatin from the fruit-eating fish, the pacu Piaractus mesopotamicus.

The fruit-eating teleost fish, the pacu Piaractus mesopotamicus (Characiformes, Characidae) is classified along with the carp and the catfish in the superorder Ostariophysi. The pacu is able to survive and grow in captive conditions feeding exclusively on carbohydrates. Hormonal polypeptides in an extract of pacu Brockmann bodies were purified to homogeneity by reversed phase HPLC and their primary structures determined by automated Edman degradation. Pacu insulin contains only two substitutions, Glu-->Asp at A15 and Thr-->Ser at B24 (corresponding to B22 in mammalian insulins) compared with carp insulin. The B-chains of both insulins contain a dipeptide extension to the N-terminus and a deletion of the C-terminal residue compared with human insulin. Pacu glucagon differs from catfish glucagon by a single substitution at position 17 (Arg-->Gln. The primary structure of the 34 amino acid residue glucagon-like peptide (GLP) differs from catfish GLP only at positions 12 (Ser-->Ala) and 33 (Pro-->Gln). In common with other teleost species, the pacu expresses two somatostatin genes. Somatostatin-14, derived from preprosomatostatin-I (PSS-I), is identical to mammalian/catfish somatostatin-14. Although pacu somatostatin-II was not identified in this study, a peptide was purified that shows 67% sequence identity with residues (1-58) of catfish preprosomatostatin-II (PSS-II). This relatively high degree of sequence similarity contrasts with the fact that catfish PSS-II shows virtually no sequence identity with the corresponding PSS-II from anglerfish (Acanthopterygii) and trout (Protoacanthopterygii). A comparison of the primary structures of the islet hormones suggest that amino acid sequences may have been better conserved within the Ostariophysi than in other groups of the taxon Euteleostei that have been studied.

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases.

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.

Purification and characterization of insulin, glucagon, and two glucagon-like peptides with insulin-releasing activity from the pancreas of the toad, Bufo marinus.

Insulin and four peptides derived from the posttranslational processing of proglucagon have been isolated in pure form from the pancreas of the cane toad, Bufo marinus. Although Bufo insulin contains 9 amino acid substitutions, compared with human insulin, all those residues that are considered to be involved in receptor-binding and in dimer and hexamer formation have been conserved. Bufo insulin was, however, more potent (4-fold) than human insulin in inhibiting the binding of [125I-Tyr-A14] insulin to the soluble full-length recombinant human insulin receptor, which is probably a consequence of the substitution (Thr --> His) at position A-8. Bufo glucagon was isolated in two molecular forms: glucagon-29 shows only one amino acid substitution (Thr29 --> Ser), compared with human glucagon; and glucagon-36 comprises glucagon-29, extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Met-Ser. The human proglucagon gene contains one copy of glucagon-like peptide (GLP)-1, a potent insulin secretogogue, and one copy of GLP-2 that is devoid of insulin-releasing activity. In contrast, two proglucagon-derived peptides with 32- and 37-amino acid residues (GLP-32 and GLP-37), displaying greater structural similarity to human GLP-1 than to GLP-2, were isolated from Bufo pancreas. Both peptides produced concentration-dependent increases in insulin release from glucose-responsive rat insulinoma-derived BRIN-BD11 cells. The threshold concentrations producing a significant (P < 0.001) effect were 10(-8) M (GLP-32) and 10(-9) M (GLP-37), and the maximum increase in the rate of insulin release produced by 10(-6) M concentrations of both peptides was approximately 5-fold.

Purification and structural characterization of insulin and glucagon from the bichir Polypterus senegalis (Actinopterygii: Polypteriformes).

The Polypteriformes (bichirs and reedfish) are a family of ray-finned fishes of ancient lineage. Insulin has been isolated from an extract of the pancreas and upper gastrointestinal tract of the bichir Polypterus senegalis and its primary structure established as A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Asp-Thr-Pro10-Cys-Ser- Leu-Tyr-Asp-Leu-Glu-Asn-Tyr-Cys20-Asn: B-chain: Ala-Ala-Asn-Arg-His-Leu-Cys-Gly-Ser-His10-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly20-Asn-Arg-Gly-Phe- Phe-Tyr-Ile-Pro-Ser-Lys30-Met. Despite the fact that Polypterus insulin contains several unusual structural features that are not found in insulins from other jawed fish (Asp at A-8, Thr at A-9, Arg at B-4, Asn at B-21, Ile at B-27, Met at B-31), all the residues in human insulin that are involved in receptor binding, dimerization, and hexamerization have been conserved. A comparison of the structures of insulins from a range of species indicates that Polypterus insulin most closely resembles paddlefish insulin II (seven amino acid substitutions). In contrast, Polypterus glucagon (His-Ser- Gln-Gly-Thr-Phe-Thr-Asn-Asp-Tyr10-Thr-Lys-Tyr- Gln-Asp-Ser-Arg-Arg-Ala-Gln20-Asp-Phe-Val-Gln- Trp-Leu-Met-Ser-Asn) most closely resembles the glucagons from the gar Lepisosteus spatula and the bowfin Amia calva (four amino acid substitutions). The data are consistent with the conclusion based on comparison of morphological characteristics that the Polypterids are the most basal living group of the Actinopterygians with evolutionary connections to both the Acipenserids and the Neopterygians.

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6 percent and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1 percent and a purification factor of 3.90 for the second, and a recovery of 76.2 percent and a purification factor of 2.36 for the last one. The overall yield was 50 percent, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84 percent pure as analyzed by HPLC.

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel.

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Purification and characterization of islet hormones (insulin, glucagon, pancreatic, polypeptide and somatostatin) from the Burmese python, Python molurus.

Insulin was purified from an extract of the pancreas of the Burmese python, Python molurus (Squamata:Serpentes) and its primary structure established as: A Chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Asn-Thr10-Cys-Ser-Leu-Tyr-Glu-Leu- Glu-Asn-Tyr-Cys20-Asn. B-Chain: Ala-Pro-Asn-Gln-His-Leu-Cys-Gly-Ser-His10-Leu-Val-Glu-Ala-Leu-Tyr- Leu-Val-Cys-Gly20-Asp-Arg-Gly-Phe-Tyr-Tyr-Ser-Pro-Arg-Ser30. With the exception of the conservative substitution Phe --> Tyr at position B25, those residues in human insulin that comprise the receptor-binding and those residues involved in dimer and hexamer formation are fully conserved in python insulin. Python insulin was slightly more potent (1.8-fold) than human insulin in inhibiting the binding of [125I-Tyr-A14] insulin to the soluble full-length recombinant human insulin receptor but was slightly less potent (1.5-fold) than human insulin for inhibiting binding to the secreted extracellular domain of the receptor. The primary structure of python glucagon contains only one amino acid substitution (Ser28 --> Asn) compared with turtle/duck glucagon and python somatostatin is identical to that of mammalian somatostatin-14. In contrast, python pancreatic polypeptide (Arg-Ile-Ala-Pro-Val-Phe-Pro-Gly-Lys-Asp10-Glu-Leu-Ala-Lys-Phe- Tyr20-Thr-Glu-Leu-Gln-Gln-Tyr-Leu-Asn-Ser-Ile30-Asn-Arg-Pro-Arg -Phe.NH2) contains only 35 instead of the customary 36 residues and the amino acid sequence of this peptide has been poorly conserved between reptiles and birds (18 substitutions compared with alligator and 20 substitutions compared with chicken).

Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells.

Lately, TNF alpha has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however, still unclear. To analyze the effects of TNF alpha in insulin-dependent diabetes mellitus (IDDM), we have generated nonobese diabetic (NOD) transgenic mice expressing TNF alpha under the control of the rat insulin II promoter (RIP). In transgenic mice, TNF alpha expression on the islets resulted in massive insulitis, composed of CD4+ T cells, CD8+ T cells, and B cells. Despite infiltration of considerable number of lymphoid cells in islets, expression of TNF alpha protected NOD mice from IDDM. To determine the mechanism of TNF alpha action, splenic cells from control NOD and RIP-TNF alpha mice were adoptively transferred to NOD-SCID recipients. In contrast to the induction of diabetes by splenic cells from control NOD mice, splenic cells from RIP-TNF alpha transgenic mice did not induce diabetes in NOD-SCID recipients. Diabetes was induced however, in the RIP-TNF alpha transgenic mice when CD8+ diabetogenic cloned T cells or splenic cells from diabetic NOD mice were adoptively transferred to these mice. Furthermore, expression of TNF alpha in islets also downregulated splenic cell responses to autoantigens. These data establish a mechanism of TNF alpha action and provide evidence that local expression of TNF alpha protects NOD mice from autoimmune diabetes by preventing the development of autoreactive islet-specific T cells.

Isolation and structural characterization of proglucagon-derived peptides, pancreatic polypeptide, and somatostatin from the urodele Amphiuma tridactylum.

The expression of the preproglucagon gene in vertebrates is markedly species- and tissue-dependent. Three peptides derived from the posttranslational processing of preproglucagon were isolated from an extract of the pancreas of the urodele Amphiuma tridactylum (threetoed amphiuma). The primary structures of the peptides indicated identity with glucagon (HSQGTFTSDY10 SKYLDNRRAQ20 DFIQWLMST), glucagon-like peptide-1 (GLP-1) (HADGTLTSDI10 SSFLEKQATK20 EFIAWLVSGR30 GRRQ), and glucagon-like peptide-2 (GLP-2) (HADGSFTSDI10 NKVLDTIAAK20 EFLNWLISTK30 VTE). Thus, in a urodele, as in the bullfrog but in contrast to the chicken and all nontetrapod species yet studied, pancreatic preproglucagon mRNA encodes a GLP-2 sequence. The amino acid sequence of glucagon has been better conserved during evolution of tetrapods (3 substitutions between amphiuma and human) than the sequences of either GLP-1 (7 substitutions) or GLP-2 (15 substitutions). Pancreatic polypeptide was also isolated from the extract and its primary structure (APKEPEHPGD10 DASPEQLEKY20 YQDLFQYIIF30 ITRPRY.NH2) indicates that the amino acid sequence of this peptide has been very poorly conserved, even among the amphibia. Amphiuma pancreatic somatostatin is identical to mammalian somatostatin-14.

Characterization of the pancreatic hormones from the Brockmann body of the tilapia: implications for islet xenograft studies.

The Brockmann body of the teleost fish, the tilapia (Oreochromis nilotica) has been considered as a potential source of islet xenograft tissue for patients with insulin-dependent diabetes. This study describes the purification from an extract of tilapia Brockmann bodies of insulin and several peptides arising from different pathways of post-translational processing of two proglucagons, two prosomatostatins and proPYY. The primary structure of tilapia insulin is similar to insulins from other teleosts (particularly the anglerfish, Lophius americanus) except that the strongly conserved glutamine residue at position 5 in the A-chain, a residue that is important in the binding of insulin to its receptor, is replaced by glutamic acid. In common with other teleosts, the tilapia Brockmann body expresses two non-allelic glucagon genes. Alternative pathways of post-translational processing lead to glucagons with 29 and 36 amino acid residues derived from proglucagon I and glucagons with 29 and 32 residues derived from proglucagon II. Glucagon-like peptides with 30 and 34 residues derived from proglucagon II were also isolated. In each case, the longer peptide is a C-terminally extended form of the shorter. Tilapia peptide tyrosine-tyrosine (PYY) was isolated in a C-terminally alpha-amidated from with 36 amino acid residues that is structurally similar (89% sequence identity) to anglerfish PYY. A 30-amino acid peptide, representing the C-terminal flanking peptide of PYY, was also isolated that shows only 53% sequence identity with the corresponding anglerfish peptide. Tilapia somatostatin-14 is identical to mammalian somatostatin but the [Tyr7, Gly10] somatostatin-containing peptide derived from prosomatostatin II contains the additional substitution (Phe11-->Leu) compared with the corresponding peptide from other teleosts.

Changes in annexin I and II levels during the postnatal development of rat pancreatic islets.

The expression patterns and the dynamic changes in content of both annexin I and annexin II in the rat pancreatic islets during postnatal development were investigated by both western blot analysis and immunohistochemistry. Immunohistochemical methods clearly demonstrated the presence of annexins I and II exclusively in pancreatic islets, while exocrine tissues were not stained by anti-annexin antibodies. Pancreatic islets were diffusely stained with no specific differences in distribution between different cell types. The expression of annexin I in pancreatic islets gradually increased with postnatal development. A developmental study of annexins I and II by western blot analysis essentially supported the results obtained by immunohistochemistry. In addition, the increasing expression of two protein tyrosine kinases, epidermal growth factor-receptor/kinase and pp60src, which phosphorylate annexin I and annexin II, respectively, and of protein kinase C, which phosphorylates both proteins, was also shown during postnatal development in rat pancreatic islets. Thus, a relationship between the expression of annexins I and II and the maturation of islet cell function is suggested.

Primary structures of peptides derived from proglucagon isolated from the pancreas of the elasmobranch fish, Scyliorhinus canicula.

Three peptides derived from the posttranslational processing of proglucagon have been isolated from the pancreas of the elasmobranch fish, Scyliorhinus canicula (European common dogfish). The peptide HSEGT FTSDY SKYMD NRRAK DFVQW LMST represents the 29 amino acid residue form of glucagon previously identified in dogfish intestine. A second component with 33 amino acid residues represents glucagon extended from its COOH-terminal region by -KRNG. The peptide HAEGT YTSDV DSLSD YFKAK RFVDS LKSY represents glucagon-like peptide (GLP). The primary structures of the GLPs from mammals have strongly conserved but a comparison of the amino acid sequences of known GLPs from different classes of fish shows that the structure of the peptide has been very poorly conserved in lower vertebrates. Only three residues (Ala2, Asp9, and Leu26) are found in the same position in all fish GLPs. A similar comparison of the primary structures of glucagons from the same species shows 13 amino acid residues in common.