Yoctomole detection of proteins using solid phase binding-induced DNA assembly

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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A receptor binding domain of mouse interleukin-4 defined by a solid-phase binding assay and in vitro mutagenesis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)49789-5 ◽

1992 ◽

Vol 267 (17) ◽

pp. 11957-11963

Author(s):

B.W. Morrison ◽

P Leder

Keyword(s):

Receptor Binding ◽

Binding Assay ◽

Solid Phase ◽

Binding Domain ◽

Interleukin 4 ◽

In Vitro Mutagenesis ◽

Receptor Binding Domain ◽

Solid Phase Binding

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Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

Canadian Journal of Microbiology ◽

10.1139/m89-063 ◽

1989 ◽

Vol 35 (3) ◽

pp. 409-415 ◽

Cited By ~ 77

Author(s):

Anthony B. Schryvers ◽

B. Craig Lee

Keyword(s):

Binding Proteins ◽

Solid Phase ◽

Bacterial Species ◽

Binding Activity ◽

Intact Cells ◽

Human Transferrin ◽

Affinity Isolation ◽

Lactoferrin Receptor ◽

Solid Phase Binding ◽

Branhamella Catarrhalis

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin–agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.Key words: iron, Neisseria, transferrin, lactoferrin, receptor.

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Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615223 ◽

1998 ◽

Vol 80 (09) ◽

pp. 418-422 ◽

Cited By ~ 31

Author(s):

Kirsty Lapan ◽

Philip Fay

Keyword(s):

Serine Protease ◽

Heavy Chain ◽

Solid Phase ◽

Activated Protein C ◽

Cross Linking ◽

Factor X ◽

Serine Protease Domain ◽

C Terminus ◽

Factor Viiia ◽

Solid Phase Binding

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

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Generation of anti-type III pneumococcal polysaccharide hybridomas from mice with an X-linked B-lymphocyte defect.

Journal of Experimental Medicine ◽

10.1084/jem.150.3.698 ◽

1979 ◽

Vol 150 (3) ◽

pp. 698-702 ◽

Cited By ~ 19

Author(s):

K R Schroer ◽

K J Kim ◽

B Prescott ◽

P J Baker

Keyword(s):

B Cell ◽

Somatic Cell ◽

B Lymphocyte ◽

Solid Phase ◽

Cell Subset ◽

Somatic Cell Hybrids ◽

Spleen Cells ◽

Type Iii ◽

Solid Phase Binding ◽

Free Antigen

(CBA/N X BALB/c male)F1 mice bear on X-linked defect making them totally unresponsive to T-independent (TI), TI-2 antigens such as type III pneumococcal polysaccharide (SSS-III). We found that somatic cell hybrids between CB nonresponder spleen cells and NS1 plasmacytoma cells secreted antibody specific for SSS-III. The solid-phase binding of such antibody was completely inhibited by the addition of free antigen (SSS-III) and the amount of antibody detected in culture fluids ranged from 10 ng/ml to 10 micrograms/ml. Eight hybridoma clones were identified; all make antibody of the IgM class. These results indicate that the X-linked defect does not result in a deletion of a B-cell subset which responds to TI-2 antigens.

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Ultrastructural Evidence for Proteoglycans Mediating an Attachment of Type VI Collagen to Banded Collagen Fibrils

Microscopy and Microanalysis ◽

10.1017/s1431927600007650 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 153-154

Author(s):

Douglas R. Keene ◽

Catherine C. Ridgway ◽

Renato V. Iozzo

Keyword(s):

Dermatan Sulfate ◽

Core Protein ◽

Solid Phase ◽

Collagen Fibrils ◽

Binding Assays ◽

Connective Tissues ◽

Type Vi Collagen ◽

Dermatan Sulfate Proteoglycan ◽

Individual Type ◽

Solid Phase Binding

Immunolocalizaton studies of type VI collagen in skin have previously demonstrated that type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into the surrounding connective tissues matrix. The purpose of this study is to determine if individual type VI collagen microfilaments might be connected to banded collagen fibrils, thereby stabilizing the network.Solid phase binding assays suggest a specific, high affinity interaction between the core protein of the dermatan sulfate proteoglycan decorin and type VI collagen, and immunocytochemical studies in fetal and neonate rabbit cornea suggest an association of decorin with type VI microfilaments. Other studies in skin and perichondrium have localized decorin to a region between the d and e bands of banded collagen fibrils. However, no direct documentation has demonstrated a specific structural interaction between type VI microfilaments and banded collagen fibrils. We, therefore, sought to determine if type VI microfilaments cross banded collagen fibrils between the “d” and “e” bands.

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Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen

Blood ◽

10.1182/blood.v88.7.2569.bloodjournal8872569 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2569-2577 ◽

Cited By ~ 48

Author(s):

S Godyna ◽

M Diaz-Ricart ◽

WS Argraves

Keyword(s):

Extracellular Matrix ◽

Vascular Injury ◽

Whole Blood ◽

Platelet Adhesion ◽

Solid Phase ◽

Blood Perfusion ◽

General Mechanism ◽

Binding Assays ◽

Solid Phase Binding ◽

Coated Surfaces

Fibulin-1 is a component of the extracellular matrix that surrounds vascular smooth muscle. This observation, along with the recent finding that fibulin-1 can bind fibrinogen (J Biol Chem 270:19458, 1995), prompted investigation into the potential role of fibulin-1 as a thrombogenic agent. In perfusion chamber assays, platelets in whole blood under flow conditions attached and spread on surfaces coated with fibulin-1. This adhesion was completely blocked by fibulin-1 antibodies. Platelets free of plasma did not attach to fibulin-1 coated surfaces; however, with the addition of fibrinogen, platelet adhesion to fibulin-1 took place. When detergent extracts of platelets were subjected to fibulin-1-Sepharose affinity chromatography, the integrin alpha IIb beta 3 was selected. Solid phase binding assays using purified components showed that integrin alpha IIb beta 3 could not bind directly to fibulin-1 but in the presence of fibrinogen the integrin bound to fibulin-1-coated surfaces. Monoclonal alpha IIb beta 3 antibodies capable of blocking its interaction with fibrinogen completely blocked platelet adhesion to fibulin-1 in both whole blood perfusion and static adhesion assays. The results show that fibulin-1 can support platelet attachment via a bridge of fibrinogen to the platelet integrin alpha IIb beta 3. When fibroblast monolayers containing extracellular matrix-incorporated fibulin-1 were used as adhesion substrates, platelet adhesion in the presence of fibrinogen could be inhibited by 30% using antibodies to fibulin-1. Following vascular injury, fibulin-1 present in the extracellular matrix of the vessel wall may therefore interact with plasma fibrinogen and promote platelet adhesion, leading to the formation of a platelet plug. Thus, fibulin-1 joins the list of matrix proteins including collagens I and IV and fibronectin that mediate platelet adhesion via a plasma protein bridge. This bridging phenomenon may represent a general mechanism by which platelets interact with exposed subendothelial matrices following vascular injury.

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Immobilized whole algal cells for solid-phase binding assays

Journal of Immunological Methods ◽

10.1016/0022-1759(86)90449-7 ◽

1986 ◽

Vol 86 (2) ◽

pp. 171-177 ◽

Cited By ~ 3

Author(s):

Paul Bubrick ◽

Leon Goldstein ◽

Asher Frensdorff

Keyword(s):

Solid Phase ◽

Binding Assays ◽

Solid Phase Binding

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Interaction between the pili of Pseudomonas aeruginosa PAK and its carbohydrate receptor β-D-GalNAc(1->4) β-D-Gal analogs

Canadian Journal of Microbiology ◽

10.1139/w97-149 ◽

1998 ◽

Vol 44 (3) ◽

pp. 307-311 ◽

Cited By ~ 2

Author(s):

Frank Schweizer ◽

Hailong Jiao ◽

Ole Hindsgaul ◽

Wah Y Wong ◽

Randall T Irvin

Keyword(s):

Pseudomonas Aeruginosa ◽

Inhibitory Concentration ◽

Binding Assay ◽

Solid Phase ◽

Hydroxyl Group ◽

Side Chain ◽

Surface Receptors ◽

Order Of Magnitude ◽

Epithelial Cell Surface ◽

Solid Phase Binding

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surface receptors. Previously, it has been shown that the pilus adhesin of P. aeruginosa PAK binds to the ganglioside asialo-GM1. In particular, it was found that the carbohydrate sequence β-D-GalNAc(1->4) β-D-Gal is the minimal carbohydrate receptor sequence of asialo-GM1. To study the binding specificity of P. aeruginosa, O-modified and N-modified sugar analogs, where each hydroxyl group was substituted either by O-methyl or O-propyl and the acetamido group was changed to a propionamido group, were synthesized. The sugar analogs were evaluated as inhibitors in a competitive solid phase binding assay. The results demonstrate that the pili of P. aeruginosa PAK accepts a variety of sugar analogs possessing the sequence β-D-GalNAc(1->4) β-D-Gal. Most sugar analogs bind with a similar order of magnitude (50% inhibitory concentration (IC50) = 60-130 μM) except for the 2-O-propyl derivative 7 (IC50 = 8 ± 4 μM) compared with an IC50 of 79 ± 18 μM for the native compound. The significant increase in binding affinity of 2-O-propyl derivative 7 suggests that improved inhibitors of adhesion may be prepared by introducing a hydrophobic side chain at the 2-position of galactose.Key words: Pseudomonas aeruginosa, pili, adhesion, carbohydrate.

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