Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies.
Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.
Cochet
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Laboratoire de Bioorganique UA CNRS 1389, Ecole Nationale Supérieure de Chimie de Paris, France.
Badet
B
B
Teillaud
J L
JL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000915:Antibody Affinity; D001667:Binding, Competitive; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005972:Glutaminase / Q000037:antagonists & inhibitors / Q000276:immunology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D007120:Immunochemistry; D007700:Kinetics; D051379:Mice; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005972:Glutaminase