Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/74779
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Type: Journal article
Title: Localization of inhibitory antibodies to the biotin domain of human pyruvate carboxylase
Author: Arpornsuwan, T.
Carey, K.
Booker, G.
Polyak, S.
Wallace, J.
Citation: Hybridoma, 2012; 31(5):305-313
Publisher: Mary Ann Liebert, Inc. Publishers
Issue Date: 2012
ISSN: 1554-0014
1557-8348
Statement of
Responsibility: 
Teerakul Arpornsuwan, Karen J. Carey, Grant W. Booker, Steven W. Polyak, and John C. Wallace
Abstract: Pyruvate carboxylase [EC 6.4.1.1] plays an important anaplerotic role in many species by catalyzing the carboxylation of pyruvate to oxaloacetate. To extend our understanding about the structure and function of pyruvate carboxylase (PC), a series of monoclonal antibodies were raised against sheep liver PC and those displaying inhibitory activity were further characterized. The binding epitopes of two monoclonal antibodies that displayed strong inhibitory activity were mapped. Six overlapping fragments of the human enzyme were expressed as thioredoxin fusion proteins in Escherichia coli and subjected to Western blot analysis. Both monoclonal antibodies (MAbs) recognized fragments encompassing the enzyme's C-terminal region, known to contain the structured biotin domain. Through deletion analysis, this domain was determined to be a minimal size of 80 amino acids. Further deletions that disrupted the conformation of the domain abolished antibody binding, indicating these antibodies recognized discontinuous epitopes. To further define the critical residues required for antibody recognition, a model of the domain was produced and an alanine scan performed on selected surface-exposed residues. Our results show that residues encompassing the biotin attachment site, but not biotin itself, are critical for the binding of both antibodies. These data provide a mechanism to explain the inhibitory activity of the antibodies.
Keywords: dough rheology; REML; PCR; relationship matrix; glutenins; puroindolines
Rights: Copyright ©2012 Mary Ann Liebert, Inc
RMID: 0020122855
DOI: 10.1089/hyb.2012.0044
Appears in Collections:IPAS publications
Molecular and Biomedical Science publications

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