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

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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Production of transferrin receptors byHistophilus ovis: three of five strains require two signals

Canadian Journal of Microbiology ◽

10.1139/w01-022 ◽

2001 ◽

Vol 47 (5) ◽

pp. 417-423 ◽

Cited By ~ 8

Author(s):

Andrew Ekins ◽

Donald F Niven

Keyword(s):

Solid Phase ◽

Iron Acquisition ◽

Isolation Procedure ◽

Transferrin Receptors ◽

Binding Assays ◽

Surface Receptors ◽

Iron Restriction ◽

Affinity Isolation ◽

Solid Phase Binding ◽

Bovine Transferrin

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.Key words: Histophilus ovis, iron acquisition, transferrins, receptors, regulation.

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Gel Entrapment of Antibody: A New Strategy for Facilitating Both Manual and Automated Radioimmunoassay

Clinical Chemistry ◽

10.1093/clinchem/19.12.1339 ◽

1973 ◽

Vol 19 (12) ◽

pp. 1339-1344 ◽

Cited By ~ 8

Author(s):

Stuart J Updike ◽

John D Simmons ◽

Douglas H Grant ◽

Judith A Magnuson ◽

Theodore L Goodfriend

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Room Temperature ◽

Solid Phase ◽

Binding Activity ◽

Angiotensin I ◽

Chromatographic Columns ◽

New Strategy ◽

Sample Application ◽

Solid Phase Binding

Abstract A technique of radioimmunoassay is presented that eliminates pipetting and centrifugation, and excludes interferences by high-molecular-weight materials from the incubation and separation steps. A solid-phase binding reagent is prepared by first entrapping antibody in polyacrylamide gel. This gel is then fragmented, sieved, dried with ethanol or lyophilized, and placed in miniature disposable chromatographic columns. Application of the sample to the intra-gel column compartment is determined by the water regain of the gel. This pipetless method of sample application depends on reproducible aliquots of dry gel particles in every column. A method for preloading radiolabeled hormone and standard hormone into the column is also described. This technique has been successfully applied to the assay of angiotensin I and insulin. Dry antibody—gel stored at room temperature for 26 months has not shown loss of binding activity.

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Isolation and characterization of galectin-1 binding proteins from human placenta

Journal of the Serbian Chemical Society ◽

10.2298/jsc0002131j ◽

2000 ◽

Vol 65 (2) ◽

pp. 131-140

Author(s):

Miroslava Jankovic

Keyword(s):

Human Placenta ◽

Binding Proteins ◽

Solid Phase ◽

Ricinus Communis ◽

Gel Filtration ◽

Isolation And Characterization ◽

Sds Page ◽

Molecular Masses ◽

Solid Phase Binding

Galectin-1 binding proteins were isolated from human placenta by affinity chromatography on a column with immobilized endogenous lectin. The molecular masses of the isolated proteins of 170, 67 and 56 kDa were estimated by gel filtration and SDS-PAGE. These proteins were characterized as galactose-containing glycoproteins, based on their reactivity with Ricinus communis agglutinin. In addition, sialylated- lacto-N-fucopentaose II was detected in the 170 kDa protein, using anti CA 19-9 monoclonal antibodies. The interaction of the isolated proteins with human placental galectin-1 was investigated by a solid phase binding assay using asialofetuin as the glycoprotein ligand. The 67 kDa and 56 kDa proteins were found to inhibit galectin-1 binding of asialofetuin, whereas the 170 kDa protein had the opposite effect. It caused an increase in the binding of asialofetuin, suggesting a positive cooperative binding.

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Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

Applied and Environmental Microbiology ◽

10.1128/aem.61.7.2738-2744.1995 ◽

1995 ◽

Vol 61 (7) ◽

pp. 2738-2744 ◽

Cited By ~ 17

Author(s):

E A Cowles ◽

H Yunovitz ◽

J F Charles ◽

S S Gill

Keyword(s):

Heliothis Virescens ◽

Binding Proteins ◽

Solid Phase ◽

Binding Assays ◽

Toxin Binding ◽

Solid Phase Binding

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Haemophilus somnus Possesses Two Systems for Acquisition of Transferrin-Bound Iron

Journal of Bacteriology ◽

10.1128/jb.186.13.4407-4411.2004 ◽

2004 ◽

Vol 186 (13) ◽

pp. 4407-4411 ◽

Cited By ~ 18

Author(s):

Andrew Ekins ◽

Fariborz Bahrami ◽

Ada Sijercic ◽

Deborah Maret ◽

Donald F. Niven

Keyword(s):

Solid Phase ◽

Phase Variation ◽

Pcr Amplification ◽

Single Component ◽

Receptor Expression ◽

Binding Assays ◽

Affinity Isolation ◽

Haemophilus Somnus ◽

Competition Binding ◽

Solid Phase Binding

ABSTRACT Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.

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Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

Biochemical Journal ◽

10.1042/bj20080234 ◽

2008 ◽

Vol 415 (1) ◽

pp. 27-33 ◽

Cited By ~ 45

Author(s):

Meghna Thakur ◽

Pradip K. Chakraborti

Keyword(s):

Protein Kinases ◽

Solid Phase ◽

Morphological Changes ◽

Binding Assays ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Peptidoglycan Biosynthesis ◽

Vitro Binding ◽

Solid Phase Binding

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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