Amin, T. and Larkins, A. and James, R.F.L. and Hirst, T.R. (1995) Generation of a Monocloal-Antibody that Recognizes the Amino-Terminal Decapeptide of the B-Subunit of Rscheichia-Coli Heat-Labile Enterotoxin - a New Probe for Studying Toxn Assemy Intermediates. Journal of Biological Chemistry, 270 (34). pp. 20143-20150. ISSN 0021-9258.
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Cholera toxin and the related Escherichia coil heat-labile enterotoxin are hexameric proteins comprising one B-subunit and five B-subunits. In this paper we report the generation and characterization of a monoclonal antibody, designated LDS47, that recognizes and precipitates in vivo assembly intermediates of the B-subunit (EtxB) of E. coil heat-labile enterotoxin. The monoclonal antibody is unable to precipitate native B-subunit pentamers, thus making LDS47 a useful probe for studying the early stages of enterotoxin biogenesis. The use of LDS47 to monitor the in vivo turnover of newly synthesized B-subunits in the periplasm off. coil demonstrated that (i) the turnover of unassembled B-subunits followed an apparent first order process and (ii) it occurred concomitantly with the assembly of native B-pentamers (k = 0.317 +/- 0.170 min(-1); t(1/2) = 2.2 min). No other proteins were co-precipitated with the newly synthesized B-subunits; a finding that implies that unassembled B-subunits do not stably associate with other periplasmic proteins prior to their assembly into a macromolecular complex. The use of overlapping synthetic peptides corresponding to the entire EtxB polypeptide demonstrated that the epitope recognized by LDS47 is located within the amino-terminal decapeptide of the B-subunit. From the x-ray structural analysis of the toxin (Sixma, T., Kalk, K., van Zanten, B., Dauter, Z., Kingma, J., Witholt, B., and Hol, W. G. J. (1993) J. Mel. Biol. 230, 890-918), this region appears to resemble a curved finger that clasps the adjacent B-subunit. Thus, this region might be expected to be exposed in the unfolded or unassembled subunit, but to become partially buried upon assembly and thus inaccessible to recognition by the monoclonal antibody.
|Subjects:||Q Science > QP Physiology (Living systems) > QP517 Biochemistry|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||P. Ogbuji|
|Date Deposited:||09 Jun 2009 08:38|
|Last Modified:||25 Jun 2012 10:35|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/19667 (The current URI for this page, for reference purposes)|
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