Showing papers by "University of Port Elizabeth published in 2009"

Ostrich MSEL-neurophysin belongs to the class of two-domain "big" neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin.

Abstract: Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV-neurophysin and a MSEL-neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a "big" neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr-derived fragments and of peptides obtained from trypsin and V8-protease digestion of the oxidized protein, this "big" neurophysin was found to contain an MSEL-neurophysin moiety (94 residues) still covalently associated with the COOH-terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL-neurophysin sequence closely resembles all known MSEL-neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV-neurophysin. It is also shown that the stretch of amino acids, linking the MSEL-neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL-neurophysin-copeptin molecule is the first "big" neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin-copeptin cleavage.

β-LIPOTROPIN: PRIMARY STRUCTURE OF THE HORMONE FROM THE OSTRICH PITUITARY GLAND

Abstract: The amino acid sequence of beta-lipotropin from the ostrich pituitary has been determined. It consists of 79 amino acids. The amino acid sequence has been determined as follows: H-(1)AlA-Leu-Pro-Pro-Ala-Ala-Met-Leu-Pro-(10)Ala-Ala-Ala-Glu-Glu-Glu-Glu-Gly-Gl u-Glu-(20)Glu-Glu-Glu-Gly-Glu-Ala-Glu-Lys-Glu-Asp-(30)Gly-Gly-Ser-Tyr-Arg-Met-A rg-His-Phe-Arg-(40)Trp-Gln-Ala-Pro-Leu-Lys-Asp-Lys-Arg-Tyr-(50)Gly-Gly-Phe-Met- Ser-Ser-Glu-Arg-Gly-Arg-(60)Ala-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-(70)Ile-Val -Lys-Ser-Ala-Tyr-Lys-Lys-Gly-(79)Gln-OH. When compared with the primary structures of other known beta-lipotropins, the sequence at the NH2-terminal, beta-melanotropin and beta-endorphin portions of the molecule exhibit considerable variability.

Purification and characterization of ostrich pancreatic secretory trypsin inhibitor

Abstract: Ostrich pancreatic secretory trypsin inhibitor was isolated and purified using acid extraction, salt fractionation. SP-Sephadex C-50 and QAE-Sephadex A-25 chromatography and RP-HPLC. The amino acid sequence of ostrich PSTI showed it is a single peptide chain containing 69 amino acid residues with the highest homology between ostrich and chicken PSTI. The molecular weight, as determined by electronspray mass spectrometry and from amino acid sequence data, is 7650 Da. The isoelectric point of ostrich PSTI was found to be 5.7. Ostrich PSTI specifically inhibited ostrich and commercial bovine trypsin with Ki values of 8.0 x 10(-9) and 2.4 x 10(-7) M, respectively, while no inhibitory effects were observed with other serine proteases.

Purification and primary structure of ostrich pancreatic polypeptide

Abstract: 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.

Complete amino acid sequence of a VLDV‐type neurophysin from ostrich differs markedly from known mammalian neurophysins

Abstract: The neurohypophyseal hormones vasopressin and oxytocin are known to be synthesized in eutherian mammals as part of larger precursors containing either MSEL- or VLDV-neurophysins. A neurophysin has been isolated from ostrich neurohypophyses and shown by partial amino acid sequence determination to be related to mammalian VLDV-neurophysin. The present report describes the complete amino acid sequence of this ostrich neurophysin containing 93 residues. This amino acid sequence, the first reported in birds, differs in a remarkable manner from its mammalian homolog. Indeed, it contains a large number of substitutions, including one insertion, distributed throughout the polypeptide chain when compared to known VLDV-neurophysins. Whereas many of these substitutions are localized inside the so-called constant region of the neurophysin, the highest variation can be found in the COOH-terminal region.

Purification and primary structure of ostrich insulin.

Abstract: 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.

Isolation and characterization of beta-lipotropin from the pituitary gland of the ostrich, Struthio camelus

Abstract: Avian beta-lipotropin (beta-LPH) was purified from adenohypophyseal glands of the ostrich Struthio camelus by a procedure involving acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography, Sephadex G-75 chromatography and paper electrophoresis (pH 6.7). The 90-amino acid peptide behaved as a single substance during polyacrylamide-gel electrophoresis, isoelectric focusing (pI of 6.0) and N-terminal analysis, the N-terminal amino acid being alanine. Ostrich beta-LPH exhibited lipolytic activity corresponding to an average minimal effective dose of 0.088 micrograms in rabbit adipose tissue.

Purification and primary structure of glucagon from ostrich pancreas splenic lobes.

Abstract: Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.

Mesotocin and vasotocin, two neurohypophysial hormones in the ostrich, Struthio camelus

Abstract: Two neurohypophysial hormones have been isolated from an avian species, the ostrich, Struthio camelus. Both have been characterized by amino acid analysis and sequence determination. The data obtained suggest that the oxytocin-like hormone is [Ile8-oxytocin] (mesotocin) and the vasopressin-like hormone is [Ile3-vasopressin] (vasotocin). Bioactivity measurements based on urinary conductivity showed vasotocin to be about five times as active as mesotocin.

Isolation and characterization of three lutropin isohormones from the pituitary gland of the ostrich (Struthio camelus).

Abstract: Avian lutropin has been isolated from pituitary glands of the ostrich (Struthio camelus) in three homogeneous forms (designated isohormones). The different homogeneous forms of ostrich lutropin were characterized physically and chemically in terms of molecular weight, electrophoretic mobility, isoelectric points, amino acid and carbohydrate composition. From these characteristics it was evident that the isohormones are very similar. The differences between these isohormones can be attributed to differences in carbohydrate composition, especially sialic acid.

Isolation and characterization of a neurophysin from ostrich neurohypophyses.

Abstract: 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.

Isolation, characterization and primary structure of two γ‐ variants from ostrich pituitary glands

Abstract: Two γ-LPH variants have been isolated from ostrich pituitary glands using acid acetone extraction, salt fractionation, ion exchange and gel permeation chromatography and HPLC. The two fractions appeared homogeneous on PAG-IEF (pI = 4.7) and both displayed an alanine N-terminal residue. Amino acid composition and fragmentation data for these two peptides are in agreement with that expected for the N-terminal 44 and 46 amino acid residues in ostrich β-LPH, with corresponding molecular masses of 4911 and 5154 respectively. The molecular mass of the smaller variant was confirmed by means of sedimentation equilibrium centrifugation to be 4717. The two variants displayed lower lipolytic potencies than the corresponding peptides from other species.

Binding and spectroscopic properties of ostrich neurophysins. Probing the role of residue 35 at the monomer-monomer interface

Abstract: Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr-35, a residue that provides a potential probe of the monomer-monomer interface and of allosteric interrelationships between this region and the binding site. Mesotocin-associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel-filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr-35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non-denaturing conditions. Reaction of Tyr-35 of the mesotocin-associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr-35 to an unusual 237 nm band in the mesotocin-associated protein. Small shifts in the 350 nm CD band of nitrated Tyr-35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr-35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin-associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.

Amino acid sequence and properties of vasopressin-associated elephant neurophysin

Abstract: The primary structure of an elephant neurophysin, homologous to vasopressin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer-monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tyr-75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and/or hydrogen bonding. The Tyr-75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr-49, distant from the monomer-monomer interface, but adjacent to the peptide-binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75-78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization-mediated conformational changes that alter the binding site in the unliganded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterisation of a novel peptide from ostrich adenohypophyses.

Abstract: A novel peptide has been isolated from ostrich pituitary glands using acid acetone extraction, salt fractionation, ion exchange and gel permeation chromatography and preparative paper electrophoresis. The homogeneous fraction contained a large proportion of hydrophobic amino acids apparently concentrated in a portion of the polypeptide. An amino-terminal isoleucine and carboxyl-terminal glutamine were found. The molecular weight was determined as 15 024 (ultracentrifugation) and 16 185 (amino acid analysis). A single intra-molecular disulfide bond was determined. The isoelectric point was 6.5. A possible role as part of a hormone precursor is suggested.