scholarly journals Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies

1972 ◽  
Vol 126 (1) ◽  
pp. 163-169 ◽  
Author(s):  
H Keiser ◽  
H J. Shulman ◽  
J I. Sandson
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Additional Species ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Caesium Chloride ◽  
Cartilage Proteoglycan ◽  
Core Proteins ◽  
Specific Antigens ◽  
Species Specific

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein–polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.

scholarly journals A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronate-substituted Sepharose gel

1981 ◽  
Vol 199 (2) ◽  
pp. 297-305 ◽  
Author(s):  
A Tengblad
Keyword(s):  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Constants ◽  
Minimum Size ◽  
Gel Chromatography ◽  
Binding Region ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Number Of Binding Sites

The hyaluronate-binding proteins from bovine nasal cartilage, i.e. the hyaluronate-binding region of the proteoglycan and the link protein, were labelled with 125I and separated from each other by gel chromatography. The proteins were characterized by molecular-weight determinations and their purity was established by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and immunodiffusion. The binding properties of the two proteins by hyaluronate-substituted Sepharose gel were compared. It was found that both proteins behaved similarly. They bound with the same efficiency to the gel, they showed the same time course of binding, had slightly different pH optima for binding and both proteins had a decreasing affinity for the gel with increasing ionic strength. The binding to the gel could be inhibited by soluble hyaluronate, and the minimum size of a hyaluronate oligosaccharide required for inhibition was in both cases a decasaccharide (only even-numbered oligosaccharides were tested). The proteins did not show any co-operative binding in the system tested, which could be explained by the large number of binding sites in the hyaluronate-substituted gel. Binding constants for the protein-hyaluronate interaction were estimated. A value of 1.3 x 10(7) M-1 was obtained for the hyaluronate-binding region of the proteoglycan, in agreement with literature data. The corresponding value for the link protein was 0.7 x 10(7) M-1.

scholarly journals The origin of human cartilage proteoglycan link-protein heterogeneity and fragmentation during aging

1985 ◽  
Vol 232 (3) ◽  
pp. 805-812 ◽  
Author(s):  
J S Mort ◽  
B Caterson ◽  
A R Poole ◽  
P J Roughley
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Link Protein ◽  
Cartilage Proteoglycan ◽  
Disulphide Bonds ◽  
Single Link ◽  
Chemical Deglycosylation

Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.

scholarly journals Studies on the subunits of human myeloperoxidase

1984 ◽  
Vol 222 (3) ◽  
pp. 701-709 ◽  
Author(s):  
R L Olsen ◽  
C Little
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Molecular Mass ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Additional Species ◽  
Main Protein

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

scholarly journals Cloning and Expression of a DNA Sequence Encoding a 41-Kilodalton Cryptosporidium parvum Oocyst Wall Protein

1999 ◽  
Vol 6 (6) ◽  
pp. 912-920 ◽  
Author(s):  
Mark C. Jenkins ◽  
Jim Trout ◽  
Charles Murphy ◽  
James A. Harp ◽  
Jim Higgins ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Recombinant Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cloning And Expression ◽  
Wall Material ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Oocyst Wall ◽  
Species Specific

ABSTRACT This study was conducted to produce a recombinant species-specific oocyst wall protein of Cryptosporidium parvum. Antigens unique to C. parvum were identified by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of oocyst proteins from several different Cryptosporidiumspecies. Antiserum was then prepared against a 41-kDa antigen unique toC. parvum and used to identify a recombinant DNA clone, designated rCP41. Expression of CP41 mRNA in C. parvumoocysts was confirmed by reverse transcriptase PCR (RT-PCR). Although the CP41 sequence was shown by PCR to be present in the genome ofC. baileyi, CP41 mRNA was not detected in this species by RT-PCR. Immunofluorescence staining with antiserum against recombinant CP41 detected native CP41 antigen on the surface of C. parvum oocysts but failed to detect CP41 on C. baileyi oocysts. Immunoelectron microscopy demonstrated that native CP41 was distributed unevenly on the C. parvumoocyst surface and was associated with amorphous oocyst wall material. In an enzyme-linked immunosorbent assay, purified rCP41 performed as well as native C. parvum oocyst protein in measuring the serological responses of young calves and adult cows to experimental and natural C. parvum infections. These results indicate that recombinant CP41 antigen may have potential in the immunodiagnosis of cryptosporidiosis.

scholarly journals Expression and Characterization of Streptococcal rgp Genes Required for Rhamnan Synthesis in Escherichia coli

2002 ◽  
Vol 70 (6) ◽  
pp. 2891-2898 ◽  
Author(s):  
Yukie Shibata ◽  
Yoshihisa Yamashita ◽  
Kazuhisa Ozaki ◽  
Yoshio Nakano ◽  
Toshihiko Koga
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polymerization Process ◽  
Streptococcus Sobrinus ◽  
E Coli ◽  
Genes Encoding ◽  
Group A ◽  
Specific Antigens

ABSTRACT Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.

scholarly journals A study of equilibrium binding of link protein to hyaluronate

1983 ◽  
Vol 213 (2) ◽  
pp. 445-450 ◽  
Author(s):  
M Lyon ◽  
I A Nieduszynski
Keyword(s):  
Dissociation Constants ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Molecule ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Link Protein ◽  
Equilibrium Binding ◽  
Femoral Head Cartilage

Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.

scholarly journals Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

2003 ◽  
Vol 10 (3) ◽  
pp. 383-387 ◽  
Author(s):  
Masahiro Yoneda ◽  
Takao Hirofuji ◽  
Noriko Motooka ◽  
Koji Nozoe ◽  
Kayoko Shigenaga ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gel Electrophoresis ◽  
Early Onset ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Healthy Control ◽  
Specific Antigens

ABSTRACT Bacteroides forsythus is one of the important periodontopathic bacteria, and this microorganism is known to have an S-layer outside the outer membrane. The S-layer-like antigens were recently isolated from B. forsythus, and they were found to be 270- and 230-kDa proteins in the envelope fraction. In this study, these proteins were confirmed to be specific to B. forsythus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they were clearly recognized by sera from patients with adult and early-onset periodontitis in Western immmunoblot analysis. We compared the immunoglobulin G (IgG) responses against the purified S-layer-like antigen by enzyme-linked immunosorbent assay. IgG responses against this antigen were low in healthy control subjects, but they were significantly higher in subjects with adult and early-onset periodontitis. Together with the fact that the IgG responses against the crude extract of B. forsythus did not rise significantly in patients with periodontitis, S-layer-like proteins are considered to be specific antigens of B. forsythus and may play an important role in the progression of periodontitis.

scholarly journals Protein Profile Study of Some Nigerian Sesame (Sesamum indicum L.) Accessions

2015 ◽  
Vol 3 (2) ◽  
pp. 322-329 ◽  
Author(s):  
Gbenga Olorunshola Alege
Keyword(s):  
Genetic Diversity ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sesamum Indicum ◽  
Genomic Changes ◽  
Sds Page ◽  
Sesame Genotypes ◽  
Species Specific ◽  
Total Seed

This study was carried out to investigate the genetic diversity among 23 sesame (Sesamum indicum L.) accessions obtained from different agro-ecological localities from 10 different states across 4 geopolitical zones in Nigeria using evidence from Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE). Total seed protein of the studied plants resolved on 12% SDS-PAGE showed variations in numbers and intensity of bands among the different sesame accessions. Thirteen (13) major bands were recorded in this study. Lack of unique band and presence of common band (band 7) among the 23 studied sesame accessions indicate some levels of genetic affinity and evidence of common evolutionary origin of the sesame genotypes. This band can therefore be tagged as species specific band for discriminating Sesamum indicum. Cluster analysis grouped the 23 sesame genotypes into two clusters with similarity coefficient ranging from 0.42 to 0.96 which indicates existence of genetic diversity; therefore there is ample opportunity for improving the 23 sesame genotypes. Variations in protein bands observed among the 23 studied plants could be attributed to genomic changes taken place during species diversification. It can be concluded that genetic diversity existed among Nigerian sesame for the improvement of characters of interest. Accessions 9 (YOL), 15(OTT), 22 (OFF) and 23 (JAL) are therefore recommended for used in future breeding programs for the development of improved sesame varieties.Int J Appl Sci Biotechnol, Vol 3(2): 322-329 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12734

scholarly journals Variant specific antigens of Trypanosoma brucei exist in solution as glycoprotein dimers

1981 ◽  
Vol 193 (2) ◽  
pp. 647-650 ◽  
Author(s):  
C A Auffret ◽  
M J Turner
Keyword(s):  
Trypanosoma Brucei ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Specific Antigens

Purified variant specific antigens of Trypanosoma brucei were shown to exist in solution as dimers, and occasionally as higher oligomers, as judged by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after treatment with bifunctional cross-linking reagents.

scholarly journals The organ-specificity of ferritin in human and horse liver and spleen

1973 ◽  
Vol 131 (1) ◽  
pp. 51-59 ◽  
Author(s):  
R. R. Crichton ◽  
J. A. Millar ◽  
R. L. C. Cumming ◽  
C. F. A. Bryce
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Organ Specificity ◽  
Subunit Molecular Weight ◽  
Electrophoretic Mobilities ◽  
A Value ◽  
Horse Liver ◽  
Normal Human Liver ◽  
Quaternary Structures ◽  
Species Specific

1. Ferritin was isolated from human and horse spleen and liver, and apoferritin prepared therefrom. 2. The electrophoretic mobilities of the four apoferritins were determined on polyacrylamide gels and on cellulose acetate strips, and all found to be equal. 3. Homologous ferritins share reactions of identity in immunodiffusion experiments, whereas heterologous ferritins show only partial identity. 4. The subunit molecular weight of each of the apoferritins was determined by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate and by chromatography on agarose columns in 6m-guanidine–HCl. A value of approx. 18500 was found in all cases. The proteins all had sedimentation coefficients of 17–18S. It thus seems that they have identical quaternary structures. 5. The amino acid compositions of the proteins revealed distinct differences both between organs and between species. This was confirmed by analysis of the tryptic peptide patterns, where it was found that about one-third of the peptides were common to the four proteins and the other two-thirds varied from protein to protein. 6. It is concluded that the apoferritins present in the liver and spleen of human and horse are both organ- and species-specific. 7. The apoferritin isolated from the liver of a patient with idiopathic haemochromatosis was identical with normal human liver apoferritin by the criteria described above.

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