02295nas a2200217   4500008004100000022001400041245016700055210006900222260007600291300001200367490000700379520140400386653002001790653002501810653003001835653003101865100001301896700002101909700001901930856012801949       2005                        eng d  a1087-057100aDevelopment of a simple assay protocol for high-throughput screening of mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors0 aDevelopment of a simple assay protocol for highthroughput screen  a2455 TELLER RD, THOUSAND OAKS, CA 91320 USAbSAGE PUBLICATIONS INCcOCT  a725-7290 v103 a<p>Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a &gt; 90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the K-m of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The K-m for ADP, arsenate, and Mn2+, were 2 mu M, 5 mu M, and 25 mu M, respectively. When the components were adjusted according to their K-m values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-mu l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.</p>10aBeckman Coulter10aGlutamine synthetase10ahigh-throughput screening10aMycobacterium tuberculosis1 aSingh, U1 aPanchanadikar, V1 aSarkar, Dhiman  uhttps://library.ncl.res.in/content/development-simple-assay-protocol-high-throughput-screening-mycobacterium-tuberculosis-0