scholarly journals Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 156
Author(s):  
Zhiliang Hei ◽  
Meihui Zhao ◽  
Yingying Tian ◽  
Hong Chang ◽  
Xuanri Shen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gadus Morhua ◽  
Bone Growth ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Isolation And Characterization

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. β-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

scholarly journals Unraveling the Function of the Rhodospirillum rubrum Activator of Polyhydroxybutyrate (PHB) Degradation: the Activator Is a PHB-Granule-Bound Protein (Phasin)

2004 ◽  
Vol 186 (8) ◽  
pp. 2466-2475 ◽  
Author(s):  
Rene Handrick ◽  
Simone Reinhardt ◽  
Daniel Schultheiss ◽  
Thomas Reichart ◽  
Dirk Schüler ◽  
...  
Keyword(s):  
Surface Layer ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rhodospirillum Rubrum ◽  
Ralstonia Eutropha ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Patterns

ABSTRACT Efficient hydrolysis of native poly(3-hydroxybutyrate) (nPHB) granules in vitro by soluble PHB depolymerase of Rhodospirillum rubrum requires pretreatment of nPHB with an activator compound present in R. rubrum cells (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). Edman sequencing of the purified activator (17.4 kDa; matrix-assisted laser desorption ionization—time of flight mass spectrometry) revealed identity to a hypothetical protein deduced from a partially sequenced R. rubrum genome. The complete activator gene, apdA (activator of polymer degradation), was cloned from genomic DNA, expressed as a six-His-tagged protein in recombinant Escherichia coli (M r, 18.3 × 103), and purified. The effect of ApdA on PHB metabolism was studied in vitro and in vivo. In vitro, the activity of the activator could be replaced by trypsin, but recombinant ApdA itself had no protease activity. Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein patterns of trypsin- and ApdA-treated nPHB granules isolated from different PHB-accumulating bacteria showed that trypsin activated nPHB by removing proteins of the surface layer of nPHB regardless of the origin of nPHB, but ApdA bound to and interacted with the surface layer of nPHB in a nonproteolytic manner, thereby transforming nPHB into an activated form that was accessible to the depolymerase. In vivo, expression of ApdA in E. coli harboring the PHB biosynthetic genes, phaCBA, resulted in significant increases in the number and surface/volume ratio of accumulated PHB granules, which was comparable to the effect of phasin proteins, such as PhaP in Ralstonia eutropha. The amino acid sequence of ApdA was 55% identical to the amino acid sequence of Mms16, a magnetosome-associated protein in magnetotactic Magnetospirillum species. Mms16 was previously reported to be a GTPase with an essential function in magnetosome formation (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276:48183-48188, 2001). However, no GTPase activity of ApdA could be demonstrated. We obtained evidence that Mms16 of Magnetospirillum gryphiswaldense can functionally replace ApdA in R. rubrum. Fusions of apdA and mms16 to gfp or yfp were functionally expressed, and both fusions colocalized with PHB granules after conjugative transfer to R. rubrum. In conclusion, ApdA in vivo is a PHB-bound, phasin-like protein in R. rubrum. The function of Mms16 in magnetotactic bacteria requires further clarification.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

Abstract The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals Heterologous Expression and Characterization of A Novel Ochratoxin A Degrading Enzyme, N-acyl-L-amino Acid Amidohydrolase, from Alcaligenes faecalis

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 518
Author(s):  
Honghai Zhang ◽  
Yunpeng Zhang ◽  
Tie Yin ◽  
Jing Wang ◽  
Xiaolin Zhang
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Degrading Enzyme ◽  
Sds Page ◽  
Ochratoxin Α ◽  
Acid Amidohydrolase

Ochratoxin A (OTA) is a well-known, natural contaminant in foods and feeds because of its toxic effects, such as nephrotoxicity in various animals. Recent studies have revealed that Alcaligenes faecalis could generate enzymes to efficiently degrade OTA to ochratoxin α (OTα) in vitro. In an effort to obtain the OTA degrading mechanism, we purified and identified a novel degrading enzyme, N-acyl-L-amino acid amidohydrolase (AfOTase), from A. faecalis DSM 16503 via mass spectrometry. The same gene of the enzyme was also encountered in other A. faecalis strains. AfOTase belongs to peptidase family M20 and contains metal ions at the active site. In this study, recombination AfOTase was expressed and characterized in Escherichia coli. The molecular mass of recombinant rAfOTase was approximately 47.0 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a wide temperature range (30–70 °C) and pH adaptation (4.5–9.0) and the optimal temperature and pH were 50 °C and 6.5, respectively.

scholarly journals Isolation and characterization of the native glycoprotein from pig small-intestinal mucus

1981 ◽  
Vol 195 (1) ◽  
pp. 267-275 ◽  
Author(s):  
M Mantle ◽  
A Allen
Keyword(s):  
Nucleic Acid ◽  
Analytical Ultracentrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intestinal Mucus ◽  
Isolation And Characterization ◽  
Average Maximum ◽  
Small Intestinal ◽  
Covalently Bound

Glycoprotein from pig small-intestinal mucus was isolated free of non-covalently bound protein and nucleic acid with a yield of over 60%. No non-covalently bound protein could be detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or by equilibrium centrifugation in a density gradient of CsCl with 4 M-guanidinium chloride. The intrinsic viscosity and reduced viscosity of the glycoprotein preparations rose with the removal of non-covalently bound protein and nucleic acid from the glycoprotein, evidence that non-covalently bound protein does not contribute to the rheological properties of the glycoprotein in the mucus. The pure glycoprotein, in contrast with impure preparations, gelled at the same concentration of glycoprotein as that present in the gel in vivo. The glycoprotein was a single component, as judged by gel filtration and analytical ultracentrifugation. The distribution of sedimentation coefficients was polydisperse but unimodal with an s025,w of 14.5S and a molecular weight of 1.72 X 10(6). The chemical composition of the glycoprotein was 77% carbohydrate and 21% protein, 52% of which was serine, threonine and proline. The glycoprotein had a strong negative charge and contained 3.1% and 18.3% by weight ester sulphate and sialic acid respectively. The molar proportion of N-acetylgalactosamine was nearly twice that of any of the other sugars present, the glycoprotein had A and H blood-group activity and the average maximum length of the carbohydrate chains was deduced to be six to eight sugar residues.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

2019 ◽  
Author(s):  
M Karunakaran ◽  
Vivek C Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S K Das ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heparin Binding ◽  
Sds Page ◽  
Significant Difference ◽  
Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

scholarly journals A T cell receptor V alpha domain expressed in bacteria: does it dimerize in solution?

1996 ◽  
Vol 184 (4) ◽  
pp. 1251-1258 ◽  
Author(s):  
D Plaksin ◽  
S Chacko ◽  
P McPhie ◽  
A Bax ◽  
E A Padlan ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Receptor ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Gel Chromatography ◽  
Mhc Class I Molecule

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR V alpha from a hybridoma with specificity for the human immunodeficiency virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2Dd. This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-V alpha 2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of beta sheet. Conditions for crystallization were established, and at least two crystal geometries were observed: hexagonal bipyramids that failed to diffract beyond approximately 6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the V alpha domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at a concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some V alpha domains have little tendency to form homodimers or multimers.

scholarly journals Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2283-2290 ◽  
Author(s):  
H Hoogendoorn ◽  
CH Toh ◽  
ME Nesheim ◽  
AR Giles
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Metal Ion ◽  
Protein C ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Molecular Weight Complex

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

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