scholarly journals Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions

1986 ◽  
Vol 234 (1) ◽  
pp. 43-48 ◽  
Author(s):  
E J Bergey ◽  
M J Levine ◽  
M S Reddy ◽  
S D Bradway ◽  
I Al-Hashimi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Gel Filtration ◽  
Cross Linking ◽  
Oral Streptococci ◽  
Bacterial Surface ◽  
Acetylneuraminic Acid ◽  
Streptococcus Sanguis ◽  
Surface Examination ◽  
Inhibition Studies

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.

scholarly journals Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

2005 ◽  
Vol 73 (7) ◽  
pp. 3851-3859 ◽  
Author(s):  
Daniel Müller ◽  
Inga Benz ◽  
Damini Tapadar ◽  
Christian Buddenborg ◽  
Lilo Greune ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Translocation ◽  
Gel Filtration ◽  
Analytical Techniques ◽  
Cross Linking ◽  
Secretion Systems ◽  
Bacterial Surface ◽  
Passenger Proteins

ABSTRACT Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal α-domain (“passenger”) fused to a C-terminal β-domain (“translocator”). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted “hairpin model” the passenger proteins are translocated through a channel formed by the β-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six β-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.

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 Rearrangement of immune complexes in glomeruli leads to persistence and development of electron-dense deposits.

1983 ◽  
Vol 157 (5) ◽  
pp. 1516-1528 ◽  
Author(s):  
M Mannik ◽  
L Y Agodoa ◽  
K A David
Keyword(s):  
Immune Complexes ◽  
Gel Electrophoresis ◽  
Immunofluorescence Microscopy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Linking ◽  
Dense Deposits ◽  
Short Period ◽  
Initial Deposition

Covalently, cross-linked immune complexes were prepared with multivalent 2-nitro-4-azidophenyl X human serum albumin (NAP X HSA) and antibodies to NAP at five times antigen excess. After purification with gel filtration, affinity chromatography with antigen-agarose column, and addition of the hapten, 9.5% of the antibodies dissociated from the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. After injection of these cross-linked immune complexes into mice, glomeruli stained for the complexes by immunofluorescence microscopy for only a few hours and electron-dense deposits were not detected. In contrast, when the same immune complexes with comparable lattice but without covalent cross-linking were administered to a second group of mice, the initial deposition by immunofluorescence was comparable and then increased to extensive deposits that persisted to 96 h. In this second group of mice extensive electron-dense deposits evolved. These observations supported the conclusion that the immune complexes initially deposited from circulation must undergo rearrangement to persist and to form electron-dense deposits in glomeruli. The covalently cross-linked immune complexes existed in glomeruli only for a short period of time since these complexes could not rearrange.

Purification and biochemical characterization of acetylcholinesterase (AChE) from the excretory/secretory products of Trichostrongylus colubriformis

Parasitology ◽  
1994 ◽  
Vol 108 (5) ◽  
pp. 579-586 ◽  
Author(s):  
G. Griffiths ◽  
D. I. Pritchard
Keyword(s):  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Ache Inhibition ◽  
Trichostrongylus Colubriformis ◽  
Excess Substrate ◽  
Secretory Products ◽  
Inhibition Studies

SummaryAcetylcholinesterase (AChE) has been purified from the excretory/secretory (ES) products of Trichostrongylus colubriformis (using edrophonium chloride linked to epoxy-activated Sepharose) with yields of 40–50%. Purity was confirmed by polyacrylamide gel electrophoresis (using silver [protein] and Karnovsky [activity] stains) and measurement of specific AChE activity. Further analysis of the purified fractions by gel filtration and sucrose density gradient techniques revealed the existence of 2 forms of hydrophilic AChE (Mr 189 and 80 kDa). From the data we deduce these to be the globular monomer and dimer, G1 and G2 forms of AChE. Inhibition studies using BW284C51, iso-OMPA and excess substrate, along with substrate specificity studies, show both forms to be true acetylcholinesterases. We are currently assessing the protective immunogenicity of purified AChE in sheep.

scholarly journals Identification of Hemin-binding Protein of Oral Streptococci via Electrophoresis in SDS Polyacrylamide Gel

2019 ◽  
pp. 1-5
Author(s):  
Eun Jeong Kim ◽  
Si Young Lee
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Oral Streptococci ◽  
Streptococcus Sobrinus ◽  
Streptococcus Sanguis ◽  
Agarose Beads ◽  
Sds Page ◽  
Streptococcus Rattus

Background and Objectives: It has been reported that hemin binding proteins are involved in the mechanism of obtaining iron in some bacteria. Oral streptococci in the dental plaque are assumed to acquire iron through hemin or hemin compounds. The aim of this study was to identify the presence of a protein (hemin binding protein) involved in the hemin binding mechanism of oral streptococci. Methodology: In this study, we investigated the presence of proteins involved in hemin binding of oral streptococci through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis using hemin-agarose beads. Results: As a result of SDS-PAGE analysis, similar or different sizes of hemin binding protein bands were observed depending on the strains belonging to streptococci. The molecular weight of hemin binding protein in Streptococcus gordonii, Streptococcus rattus, Streptococcus sobrinus, Streptococcus sanguis and Streptococcus oralis were approximately 95 kDa, 43 kDa, 43 kDa, 39 kDa, and 39 kDa, respectively. Conclusion: In this study, the presence of hemin binding protein in streptococci was confirmed and the proteins involved in hemin binding in different species of oral streptococci may be different.

scholarly journals Preparation and characterization of chemically defined oligomers of rabbit immunoglobulin G molecules for the complement binding studies

1980 ◽  
Vol 187 (3) ◽  
pp. 767-774 ◽  
Author(s):  
J K Wright ◽  
J Tschopp ◽  
J C Jaton
Keyword(s):  
Immunoglobulin G ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Linking ◽  
Binding Studies ◽  
Electrophoretic Mobilities ◽  
Rabbit Igg ◽  
Linker Molecules

Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at −70 degrees C.

scholarly journals Purification and Characterization of ?-N-Acetylgalactosaminidase from Hilsha ilisha

2017 ◽  
Vol 9 (2) ◽  
pp. 235-246
Author(s):  
M. H. Rashid ◽  
A. H. M. K. Alam ◽  
J. Uddin ◽  
N. Karim ◽  
M. G. Sadik
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Ammonium Sulphate Precipitation ◽  
Heat Stable ◽  
Optimum Ph ◽  
Km Value ◽  
C 50 ◽  
Inhibition Studies

The objective of this study was to purify and characterize the alpha-N-acetylgalactosaminidase (?-GalNAcase) from hilsha fish, Hilsha ilisha. Digestive organ of hilsha fish was found to contain a large amount of ?-GalNAcase in compared with the other tissues examined. ?-GalNAcase was purified from the crude extract of hilsha fish by ammonium sulphate precipitation, Sephadex G-200 gel filtration, and SP-Sephadex C-50 column chromatography. The purified enzyme gave a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis and exhibited a molecular mass of 48 kDa. The final preparation of ?-GalNAcase showed 3.02% ?-galactosidase activity. The enzyme had an optimum pH of 4.0 and was found to be quiet heat stable at 37°C. Inhibition studies with metal ions demonstrated that the enzyme was highly inhibited by silver and mercury ions. Both N-acetylgalactosamine and galactose affect the enzyme activity. Kinetic studies with the enzyme showed that the KM value for p-nitrophenyl-?-N-acetylgalactosaminide substrate was 3.31 mM and the Vmax value was 35.04 unit/mg.

scholarly journals Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin

1989 ◽  
Vol 263 (2) ◽  
pp. 417-423 ◽  
Author(s):  
V Singh ◽  
M R Sairam
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Cross Linking ◽  
Carrier Protein ◽  
Ribosome Inactivating Protein ◽  
Reverse Phase ◽  
Specific Radioimmunoassay ◽  
Cross Linker

Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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