This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Landais, S. Articles by Sakamoto, H. Search for Related Content PubMed PubMed Citation Articles by Landais, S. Articles by Sakamoto, H.

 Previous Article  |  Next Article 

Journal of Bacteriology, February 1999, p. 833-840, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Immunochemical Analysis of UMP Kinase from Escherichia coli

Stéphanie Landais,¹ Pierre Gounon,² Christine Laurent-Winter,¹ Jean-Claude Mazié,³ Antoine Danchin,⁴ Octavian Bârzu,¹ and Hiroshi Sakamoto^1,*

Laboratoire de Chimie Structurale des Macromolécules,¹ Station Centrale de Microscopie Electronique,² Laboratoire d'Ingénierie des Anticorps,³ and Unité de Régulation de l'Expression Génétique,⁴ Institut Pasteur, 75724 Paris Cedex 15, France

Received 3 August 1998/Accepted 17 November 1998

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

---

^* Corresponding author. Mailing address: Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 (1) 40 61 37 78. Fax: 33 (1) 45 68 84 05. E-mail: hiroshi{at}pasteur.fr.

---

Journal of Bacteriology, February 1999, p. 833-840, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Watt, R. M., Wang, J., Leong, M., Kung, H.-f., Cheah, K. S.E., Liu, D., Danchin, A., Huang, J.-D. (2007). Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes. Nucleic Acids Res 35: e37-e37 [Abstract] [Full Text]  
• Evrin, C., Straut, M., Slavova-Azmanova, N., Bucurenci, N., Onu, A., Assairi, L., Ionescu, M., Palibroda, N., Barzu, O., Gilles, A.-M. (2007). Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria. J. Biol. Chem. 282: 7242-7253 [Abstract] [Full Text]  
• Sakamoto, H., Landais, S., Evrin, C., Laurent-Winter, C., Barzu, O., Kelln, R. A. (2004). Structure-function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria. Microbiology 150: 2153-2159 [Abstract] [Full Text]