Purification and primary structure of ostrich insulin

Insulin has been isolated from ostrich pancreas by a procedure of acid ethanol extraction, adsorption onto SP-Sephadex, gel permeation chromatography and HPLC. The primary structure of the ostrich insulin is identical to that reported for the chicken hormone. The isoelectric point as determined by polyacrylamide gel isoelectric focusing was significantly higher than that of the bovine hormone.     

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.     

The isolation and characterization of pepsinogens from the proventriculus of the ostrich Struthio camelus

Three pepsinogens were isolated and purified from the proventriculus of the ostrich Struthio camelus, by a combination of chromatography steps on DEAE-cellulose, Sephadex G-100 and Hydroxylapatite. The purified pepsinogens manifested peptic activity towards haemoglobin as substrate after activation, but resembled chicken pepsinogens in that they appeared to lose their potential peptic activities during storage. All three pepsinogens contained glycine as N-terminal amino acid, but differed in their overall amino acid compositions. The pH and temperature optima of the activated pepsinogens were determined, as well as their molecular weights.     

Chicken glucagon. Isolation and amino acid sequence studies.

Glucagon was isolated from a side fraction generated during the preparation of insulin and the new pancreatic peptide, avian pancreatic polypeptide from chicken pancreas. The immunological and biological properties are similar to those of beef-pork glucagon. The amino acid composition of chicken glucagon indicates that it contains 1 more serine residue than the porcine hormone and 1 less aspartic acid (asparagine) residue. Thus, chicken glucagon appears to be identical with turkey glucagon.     

Purification of duck growth hormone and cloning of the complementary DNA

Duck growth hormone (GH) was isolated and purified from duck pituitaries by salt precipitation and HPLC on reverse-phase C18 columns. The duck GH was homogeneous as shown by SDS-polyacrylamide gel electrophoresis with a molecular weight of 22,000. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length duck GH cDNA contains 820 nucleotide pairs with an open reading frame coding for the precursor form duck GH of 216 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with Phe as the first residue in mature duck GH preceded by a 27-residue hydrophobic signal peptide. The duck GH is almost completely homologous to the chicken GH, with only three conservative substitutions (Ser for Thr, His for Tyr and Lys for Arg) and one deletion (Ala) in the duck GH sequence. Comparison of amino-acid sequence of duck GH with that of various species reveals 56%, 73% and 40% homologies with GHs of human, rat and salmon, respectively.     

The purification and characterization of rabbit placental lactogen.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.     

Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae

A proteinaceous factor that represses plasma juvenile hormone esterase activity in parasitized insect larvae has been isolated and partially characterized from last instar larvae of the armyworm Pseudaletia separata parasitized with the wasp Apantales kariyai. Purification procedures consisted of extraction with 25% ethanol, gel filtration and reversed phase high performance liquid chromatography. Plasma juvenile hormone esterase activity in Day 3 last instar larvae was repressed by 50% when larvae were injected on Days 1 and 2 with 6.5 pmol of the purified peptide, which has a molecular weight of about 4,500 Da. The application of the factor also causes more than a 2-day delay in the onset of pupation. The sequence of 23 amino acid residues at the amino terminus of the factor was determined as follows: H-Glu-Asn-Phe-Ser-Gly-Gly-Xaa-Val-Ala-Gly-Tyr-Met- Arg-Thr-Pro-Asp-Gly-Arg-Xaa-Lys-Pro-Thr-Phe-Tyr-Gln-.     

Synthesis and hormonal activity of [Tyr22] glucagon and [desHis1, Tyr22] glucagon

[Tyr22] glucagon and [desHis1, Tyr22] glucagon were synthesized by an improved solid phase procedure on a Pam-resin. The course of the synthesis was monitored by quantitative ninhydrin analysis and preview sequencing. Following cleavage by the low/high HF method the peptides were purified by ion exchange chromatography and reverse phase HPLC. The overall yield of homogeneous isolated peptide from the first amino acid was 41%. Circular dichroism measurements on dilute solutions in mixed aqueous organic solvents at pH 2, 6.9 and 9.2 showed increased beta-sheet structure relative to glucagon. [Tyr22] glucagon was a full agonist with 20-30% activity in the rabbit blood glucose assay and 10% activity in the rat liver membrane adenyl cyclase assay. [desHis1, Tyr22] glucagon had only a trace of activity in the adenyl cyclase assay (less than 0.002%) but bound to membranes in a competitive [125I] glucagon assay 1.0% as well as glucagon. The analog completely inhibited formation of cAMP by natural glucagon, with 50% inhibition at a ratio of 83:1 and pA2 = 6.7. The data are discussed in terms of models of glucagon structure in dilute solution.     

Isolation and properties of the electrophoretic components of human growth hormone by Sephadex-gel filtration and preparative polyacrylamide-gel electrophoresis

1. Human growth hormone was prepared from acetone-dried residues after extraction of gonadotrophins from pituitary glands. 2. Crude growth hormone was purified by gel filtration on Sephadex, resulting in a product that is soluble in water or 0.5% sodium chloride. It is painless on injection and shows a twofold increase in biological potency. Aggregation of growth hormone on Sephadex columns can be avoided by the addition of urea (6m) and EDTA (1mm) to the buffer. 3. Growth hormone appeared as a single component from Sephadex and ion-exchange columns and sedimented as a single boundary in the ultracentrifuge. In the circular disk electrophoresis, however, the growth hormone showed one faster and two slower-moving anionic components. 4. These components were isolated by preparative electrophoresis on polyacrylamide columns. The purified growth hormone and its three components sedimented as single boundaries with coefficients 2.62, 2.66, 2.66 and 2.83s respectively. 5. Amino acid analyses of the purified growth hormone and its components were closely related. End-group analysis of purified growth hormone and its components showed only phenylalanine at both N- and C-terminals. 6. The purified growth hormone and its components were essentially free of other pituitary hormones, but contained significant prolactin activity. The biochemical similarities among the electrophoretic components of human growth hormone and the presence of the same three components in the growth hormone prepared from a single human pituitary gland suggest polymorphism of a biologically active protein molecule.     

Methylation of glucagon, characterization of the sulfonium derivative, and regeneration of the native covalent structure

The methylation of the single methionine residue of glucagon is accomplished at a pH of 3.5 in 8 M urea with methyl iodide. The reaction product is a soluble sulfonium derivative, S-methylglucagon, which can be isolated in a highly purified form. This derivative is characterized by amino acid analysis and its effect on the adenylyl cyclase system of rat liver plasma membranes. S-Methylglucagon does stimulate the adenylyl cyclase system; however, its activity is approximately 500 times less than that observed with the native hormone. The solubility of this derivative is great enough to allow for further modifications of the molecule which can be followed at a later stage by demethylation. Demethylation of S-methylglucagon regenerates the original covalent structure and is accomplished by treatment with Cleland's reagents at a pH of 10.5. The regenerated hormone is indistinguishable from native glucagon by its amino acid composition and its ability to stimulate the adenylyl cyclase system. The entire methylation-demethylation reaction sequence has been carried out with yields that approach 75%. The technique is suitable for the isotopic enrichment of native glucagon and may well be applicable to selected other methionine-containing peptides.     

Biochemical and structural comparative study between bird and mammal pancreatic colipases

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.     

Isolation and characterization of a neurophysin from ostrich neurohypophyses

A neurophysin has been isolated from ostrich neurohypophyses using acid acetone extraction, salt fractionation and Sephadex G-75 chromatography. The crude neurophysin eluting from the Sephadex G-75 column was subjected to a) reverse-phase HPLC followed by Sephadex G-75 chromatography, b) DEAE-Sephadex A-50 chromatography or c) isoelectric focusing. The different homogeneous ostrich neurophysin fractions so obtained were compared i.t.o. amino acid composition, spectral properties, N-terminal amino acid residues and PAGE. They all revealed a single N-terminal Ala residue and displayed spectral properties (A280/A260 less than 1) which are typical of mammalian neurophysin-like polypeptides. Ultracentrifugation studies on purified ostrich neurophysin over a range of concentrations revealed a reversible concentration dependent association behaviour characterized by the presence of dimeric complexes at higher concentrations. Partial sequencing from the N-terminus revealed the molecule to be VLDV-like. The purified molecule was also submitted to CNBr fragmentation.     

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).     

Binding-site specificity of the radiolytically induced crosslinking of phenylalanine to glucagon

The gamma-radiation-induced crosslinking of phenylalanine to glucagon, mediated by OH ., has been shown to involve a limited number of binding sites on the glucagon molecule. Glucagon-phenylalanine adducts were partially separated from other radiolysis products with Sephadex gel filtration; further isolation of adducts was achieved with reverse-phase high-performance liquid chromatography (HPLC). Amino acid analysis of the isolated adducts indicates that the aromatic residues (phenylalanine and tyrosine), basic residues (histidine and lysine), and sulfur-containing residue (methionine) of glucagon are predominantly involved in crosslinking; these are essentially the same residues implicated in glucagon-glucagon crosslinking. Acid hydrolysates and chymotryptic digests of glucagon-phenylalanine adducts were examined with HPLC. The number of amino acid-phenylalanine adducts and also chymotryptic peptides observed was much greater than would have been expected based on the amino acid analysis. This observation is best accounted for by the involvement in crosslinking of radicals formed on the glucagon with more than one possible phenylalanine-derived free radical.     

Glucagon from avian pancreatic islets: radioreceptor studies.

Glucagon extracted from isolated islets of the pigeon was studied by means of Sephadex gel filtration. Radioreceptor assay, using rat liver plasma membranes and radioiodinated porcine glucagon, showed that the bulk of the activity eluted with glucagon (molecular weight 3500). Avian glucagon appeared to be less effective than porcine glucagon in inhibiting the binding of labeled porcine glucagon to rat plasma membranes.     

The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus.

1. Avian corticotropin (ACTH) was purified from both fresh and aged pituitary glands of the ostrich Struthio camelus. 2. The isolation of corticotropin in pure form involved acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography and Sephadex G-50 chromatography. 3. The hormone preparations from fresh and aged glands behaved as single substances on polyacrylamide-gel electrophoresis, and both preparations were found to consist of 39 amino acid residues, in identical molar proportions for the different amino acids. 4. The isoelectric points of the two hormone preparations were estimated to be in the range pH 8.3-8.7, indicating possible differences in amide content, and the N-terminal amino acid of both preparations appeared to be serine. 5. The hormone preparations from fresh and aged glands exhibited similar biological potencies (73 and 77 i.u./mg respectively), as measured by steroidogenesis in vitro. 6. Apart from possible differences in amide content, the corticotropin preparations obtained from fresh and aged glands appear to be indistinguishable.     

Homology of single copy and repeated sequences in chicken, duck, Japanese quail, and ostrich DNA.

The extent of reassociation of 3H-labeled repetitive or single copy DNA sequences from the chicken with excess unlabeled DNA from the duck, the Japanese quail, and the ostrich, respectively, was measured by hydroxylapatite chromatography. Chicken repetitive DNA reassociated to an equal or greater extent than chicken single copy DNA with the DNA of each of the other birds. Using an isolated subfraction of chicken repetitive DNA representing those DNA sequences common to the chicken and ostrich genomes, we determined that many repetitive DNA sequences that occur at high repetition frequency in the chicken genome have a much lower repetition frequency in ostrich DNA. The data indicate that there has been a striking change in the number of copies of many repetitive DNA sequences during avian evolution.     

The purification and biological properties of pancreatic big glucagon.

1. Big glucagon was present in extracts of ox, dog, rat and turkey pancreas, representing 10-15% of the glucagon immunoreactivity, and was shown to be of islet origin by its presence in extracts of isolated pigeon islets. 2. Big glucagon was homogeneous by immunoassay after polyacrylamide-gel electrophoresis and was more electronegative than little glucagon. 3. Big glucagon was purified from bovine pancreas, and its apparent molecular weight was estimated by gel filtration as 8200+/-9%. 4. Limited tryptic proteolysis of the molecule produced an immunoreactive component slightly smaller than little glucagon. 5. Linear dilution curves were obtained with mammalian big glucagons by using both enteroglucagon cross-reacting and 'little-glucagon-carboxyl-end-specific' antisera. 6. The half-times for the disappearance of the immunoreactivity of big and little glucagon that had been injected into the rat circulation were 6.9 and 3.2min respectively. 7. Big glucagon was approximately one-sixth as effective as little glucagon in displacing radioactive little glucagon from its liver membrane receptor. 8. Big glucagon was equipotent on a molar basis with little glucagon in the stimulation of the mouse islet adenylate cyclase, an indicator of insulinogenic activity. 9. On a molar basis, big glucagon inhibited basal liver adenylate cyclase activity to the same extent that little glucagon stimulated the enzyme. 10. Big glucagon was without effect on blood glucose concentration in the rat in doses up to 5mug/kg. 11. Big glucagon was equipotent, on a molar basis, with little glucagon in stimulating lipolysis in isolated chicken fat-cells.     

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.     

The isolation and partial characterization of trypsin from the pancreas of the ostrich Struthio camelus

Cationic trypsin was isolated and purified from the pancreas of the ostrich (Struthio camelus) by affinity chromatography on a Trasylol-Sepharose column. External activation of trypsinogen was required before trypsin could be isolated. The final preparation was homogeneous by SDS-PAGE and by sedimentation equilibrium centrifugation studies, resulting in Mr values of 24,547 and 22,091, respectively. The Mmin value obtained from amino acid analysis was 22,450. A mean sedimentation coefficient of 2S was obtained by sedimentation velocity centrifugation. Results obtained from N-terminal and amino acid analyses were similar to those from trypsins of other species. The effects of pH, temperature and inhibitors (LBTI, KBPTI and PMSF) on the tryptic activity were examined. The effect of calcium ions and enzyme concentration on the rate of self-digestion of ostrich trypsin was also investigated.