AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chandrakala, B.
Right arrow Articles by deSousa, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chandrakala, B.
Right arrow Articles by deSousa, S. M.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, March 2001, p. 768-775, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.768-775.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in Escherichia coli

B. Chandrakala, Bertha C. Elias, Upasana Mehra,dagger N. S. Umapathy,Dagger P. Dwarakanath, T. S. Balganesh, and Sunita M. deSousa*

AstraZeneca India Pvt. Ltd., Bangalore 560 003, India

Received 31 July 2000/Returned for modification 19 October 2000/Accepted 7 December 2000

We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[3H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.

* Corresponding author. Mailing address: AstraZeneca India Pvt. Ltd, 277 T. Chowdaiah Rd., Malleswaram, Bangalore 560 003, India. Phone: 91-80-334-0372. Fax: 91-80-334-0449. E-mail: sunita.desousa{at}astrazeneca.com.

dagger Present address: Telik, Inc., South San Francisco, CA 94080.

Dagger Present address: Bangalore Genei Pvt. Ltd, BDA Industrial Suburb, Peenya, Bangalore 560 058, India.

Antimicrobial Agents and Chemotherapy, March 2001, p. 768-775, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.768-775.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS

Copyright © 2001 by the American Society for Microbiology. All rights reserved.