Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80 mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS–PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries.     

Natural purified porcine gastric inhibitory polypeptide (GIP) stimulates exocrine pancreas secretion due to contamination with a cholecystokinin-like substance

The effects of purified natural gastric inhibitory polypeptide-enterogastrone III (GIP-EG III) and a fraction which is further purified by high pressure liquid chromatography (GIP-HPLC) were investigated on the endocrine and exocrine isolated perfused pancreas of rats. At the dose of 5 ng/ml used for both GIP preparations, only GIP-EG III significantly stimulated volume and amylase secretion of the exocrine pancreas. The response of insulin release to stimulation by GIP-EG III or GIP-HPLC was not significantly different. In the presence of cholecystokinin-octapeptide (CCK-8) at a concentration which gave half-maximal stimulation of amylase secretion, GIP-EG III almost doubled the response of the exocrine pancreas, whereas GIP-HPLC had no additional effect. CCK-8 alone significantly increased total insulin output under hyperglycemic conditions. We conclude that porcine GIP purified by gel chromatography contains a CCK-like substance which can be removed by further purification on high pressure liquid chromatography without affecting the insulinotropic activity. Some of the reported effects of GIP could be due to contamination.     

Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64-69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4-91.7%. The final yield of the hexapeptide was 22 micrograms (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon.     

Partial structure of a large canine cholecystokinin (CCK58): Amino acid sequence

A cholecystokinin molecule larger than any previously chemically characterized was purified from canine proximal small intestine mucosa. The purification procedure consisted of sequential steps of affinity chromatography, gel filtration, and high pressure liquid chromatography. Activity was detected and quantitated by radioimmunoassay with an antibody that recognized the carboxyl terminal sequence of porcine cholecystokinin. Microsequencing of the purified peptide revealed an amino terminal nonadecapeptide sequence (AQKVNSGEPRAHLGALLAR) not present in known cholecystokinin molecules followed by a nonadecapeptide sequence (YIQQARKAPSGRMSVIKNL) that corresponds exactly to the amino terminal sequence of porcine cholecystokinin 39 except for reversed positions of a Met and a Val residue. Based on the sequence analysis, immunoreactivity, and presence of biological activity in two bioassay systems, this peptide, tentatively named cholecystokinin 58, may be a biosynthetic precursor of the smaller forms previously characterized in gastrointestinal and brain tissues.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig

Summary Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig.

Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

C-peptide, insulin and proinsulinlike components in diabetic and nondiabetic human pancreas.

The contents of insulin and C-peptide extractable with acid alcohol from the tail of the pancreas and insulinoma were investigated, using gel filtration in seven nondiabetics including two patients with insulinoma and eight diabetics. The gel filtration patterns of both C-peptide and insulin in pancreatic extract were fairly stable even after the pancreas had been left for 14 hrs in the room temperature. In nondiabetics except cases of insulinoma the content of insulin in pancreas ranged from 1.42 to 4.56 U per gram and that of C-peptide from 8.76 to 25.63 microgram per gram wet pancreas. The proportion of proinsulinlike components (PLC) ranged from 0.01 to 2.04% of insulin plus PLC. In diabetics insulin content was low and ranged from 0 to 1.68 U per gram and that of C-peptide from 0 to 14.48 microgram per gram wet pancreas. In insulinoma, both insulin and C-peptide increased and PLC occupied 5.48 and 5.96%, respectively.     

Isolation and characterization of a new pancreatic polypeptide hormone.

A method is described for isolation, from chicken pancreas, of an avian pancreatic polypeptide which may be a new hormone. This method involves acid-alcohol extraction, gel filtration, DEAE-cellulose chromatography, and droplet countercurrent distribution. The peptide contains 36 amino acids, has a molecular weight of 4240 and the isoelectric point if pH 6 to 7. The average amount of avian pancreatic polypeptide extractable from chicken pancreas was 4 mg/100 g of pancreas. The amino acid sequence of the peptide is Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.     

Studies on cytochrome c oxidase, VII. Isolation and chemical characterization of polypeptide VII.

Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.     

Pancreatic spasmolytic polypeptide: crystallization, circular dichroism analysis, and preliminary X-ray diffraction studies.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X-ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombic I222 or I2(1)2(1)2(1) with unit cell parameters a = 54.38 A, b = 72.29 A, and c = 180.85 A. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 A. The far-UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% alpha-helix, between 10 and 20% antiparallel beta-strand, 10% parallel beta-strand, 15% turn, and 25 to 40% of other structures.     

Purification and characterization of ovine pancreatic alpha - amylase.

Ovine pancreatic amylase has been purified from pancreas homogenate by ammonium sulfate, acetone precipitation, DEAE-cellulose chromatography and finally by specific adsorption on polydextran gel. The enzyme is homogeneous and found as a single form as shown by disc electrophoresis, SDS gel electrophoresis, electrofocusing and ultracentrifugation. Its specific activity is similar to that of porcine amylase. The amino acid composition indicates a high content in aromatic and acidic amino acids as for the porcine enzyme; however the methionine and half cystine content differ widely. The N-terminal end is blocked. Also ovine amylase is glycosylated. The molecular weight (56,000-58,000) is slightly higher than for the porcine enzyme. The isoelectric point is acidic (pI = 3.2).     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.     

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.     

Isolation and partial chemical characterization of THF, a thymus hormone involved in immune maturation of lymphoid cells.

A procedure for isolation and characterization of the active principle of calf thymus is described. It consists of homogenization, ultracentrifugation and dialysis of the material. The active dialyzate is further purified by gel filtration on Sephadex G-10 and G-25 columns followed by anion exchange chromatography. The level of purification is assessed by isoelectric focusing on polyacrylamide gels. The active principle is a polypeptide of MW 3220. Determination of the amino acid composition revealed the presence of high proportion of acidic residues.     

Proteoglycans of soluble fraction of mouse mastocytoma.

Proteoglycans have been isolated from a high speed supernatant fraction of a mouse mastocytoma by procedures which should minimize alteration of the native protein-polysaccharide molecule. The methods used include in vivo labeling proteoglycans with 35S-sulfate, 3H-leucine and 3H-lysine, centrifugation of the tumor homogenate at 105,000 g, cetylpyridinium fractionation of the supernatant, and further purification of some of the fractions obtained by DEAE-cellulose column chromatography, gel filtration on Sepharose 4B and cellulose acetate electrophoresis. Two major sulfated proteoglycans were obtained, one containing keratan sulfate-like material (KSP-S), the other a heparin-like polymer (HP-S). The presence in HP-S of a compound similar to heparin was confirmed by its digestibility with flavobacterium heparinase. HP-S contained about 4 per cent protein. Glycine was the predominant amino acid, and serine did not appear to be involved in the peptide-carbohydrate linkage. The proteoglycan present in HP-S appeared to be homogeneous when examined using cellulose acetate electrophoresis. KSP-S was found to contain sialic acid and its protein content was significantly higher than that of HP-S. Glutamic and aspartic acids were the most abundant amino acids in KSP-S.     

Purification and characterization of N-acetyl-D-galactosamine-binding lectin from Falcata japonica

An N-acetyl-D-galactosamine-binding lectin from Falcata japonica seeds was purified by affinity column chromatography of N-acetyl-D-galactosamine coupled to epoxy-activated Sepharose 6B. A 1000-fold purification of lectin was obtained from the crude extracts. The purified lectin agglutinated blood group A red cells, but neither blood group B nor O red cells. Polyacrylamide gel electrophoresis of the lectin showed one diffuse band. Molecular weights of 125,000 and 117,000 were estimated by gel filtration and ultracentrifugal analysis, respectively. SDS-polyacrylamide gel electrophoresis of the lectin also showed a single band which has a molecular weight of 34,000. Therefore, the lectin molecule was estimated to be a tetramer composed of four identical non-covalently bound subunits. F. japonica lectin was a glycoprotein containing 5% total carbohydrate, and the amino acid composition was characterized by a high content of aspartic acid, serine and glycine, a low content of methionine and the absence of cysteine.     

One-step purification of mammalian deoxyribonucleases I and differences among pancreas, parotid, and pancreas-parotid (mixed) types based on species- and organ-specific N-linked glycosylation

Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme.