Novel scintillation proximity assay for measuring membrane-associated steps of peptidoglycan biosynthesis in Escherichia coli

B. Chandrakala, B. C. Elias, U. Mehra, Umapathy N Siddaramappa, P. Dwarakanath, T. S. Balganesh, S. M. DeSousa

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)768-775
Number of pages8
JournalAntimicrobial Agents and Chemotherapy
Volume45
Issue number3
DOIs
StatePublished - Mar 12 2001
Externally publishedYes

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Peptidoglycan
Escherichia coli
Membranes
Uridine Diphosphate Sugars
Enzymes
Peptidyl Transferases
Nisin
Penicillin-Binding Proteins
Bacitracin
Tunicamycin
Paper Chromatography
Wheat Germ Agglutinins
Uridine Diphosphate
Acetylglucosamine
Transferases
Anti-Infective Agents
Penicillins
Cell Survival
Lipids

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

Novel scintillation proximity assay for measuring membrane-associated steps of peptidoglycan biosynthesis in Escherichia coli. / Chandrakala, B.; Elias, B. C.; Mehra, U.; Siddaramappa, Umapathy N; Dwarakanath, P.; Balganesh, T. S.; DeSousa, S. M.

In: Antimicrobial Agents and Chemotherapy, Vol. 45, No. 3, 12.03.2001, p. 768-775.

Research output: Contribution to journalArticle

Chandrakala, B. ; Elias, B. C. ; Mehra, U. ; Siddaramappa, Umapathy N ; Dwarakanath, P. ; Balganesh, T. S. ; DeSousa, S. M. / Novel scintillation proximity assay for measuring membrane-associated steps of peptidoglycan biosynthesis in Escherichia coli. In: Antimicrobial Agents and Chemotherapy. 2001 ; Vol. 45, No. 3. pp. 768-775.
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