<?xml version="1.0" encoding="UTF-8"?><xml><records><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Singh, U</style></author><author><style face="normal" font="default" size="100%">Panchanadikar, V</style></author><author><style face="normal" font="default" size="100%">Sarkar, Dhiman</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Development of a simple assay protocol for high-throughput screening of mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors</style></title><secondary-title><style face="normal" font="default" size="100%">Journal of Biomolecular Screening</style></secondary-title></titles><keywords><keyword><style  face="normal" font="default" size="100%">Beckman Coulter</style></keyword><keyword><style  face="normal" font="default" size="100%">Glutamine synthetase</style></keyword><keyword><style  face="normal" font="default" size="100%">high-throughput screening</style></keyword><keyword><style  face="normal" font="default" size="100%">Mycobacterium tuberculosis</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">2005</style></year><pub-dates><date><style  face="normal" font="default" size="100%">OCT</style></date></pub-dates></dates><number><style face="normal" font="default" size="100%">7</style></number><publisher><style face="normal" font="default" size="100%">SAGE PUBLICATIONS INC</style></publisher><pub-location><style face="normal" font="default" size="100%">2455 TELLER RD, THOUSAND OAKS, CA 91320 USA</style></pub-location><volume><style face="normal" font="default" size="100%">10</style></volume><pages><style face="normal" font="default" size="100%">725-729</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p&gt;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 &amp;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.&lt;/p&gt;</style></abstract><issue><style face="normal" font="default" size="100%">7</style></issue><work-type><style face="normal" font="default" size="100%">Article</style></work-type><custom3><style face="normal" font="default" size="100%">&lt;p&gt;Foreign&lt;/p&gt;</style></custom3><custom4><style face="normal" font="default" size="100%">2.218</style></custom4></record><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Singh, Upasana</style></author><author><style face="normal" font="default" size="100%">Sarkar, Dhiman</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Development of a simple high-throughput screening protocol based on biosynthetic activity of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors</style></title><secondary-title><style face="normal" font="default" size="100%">Journal of Biomolecular Screening</style></secondary-title></titles><keywords><keyword><style  face="normal" font="default" size="100%">Beckman Coulter</style></keyword><keyword><style  face="normal" font="default" size="100%">biosynthetic activity</style></keyword><keyword><style  face="normal" font="default" size="100%">Glutamine synthetase</style></keyword><keyword><style  face="normal" font="default" size="100%">high-throughput screening</style></keyword><keyword><style  face="normal" font="default" size="100%">Mycobacterium tuberculosis</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">2006</style></year><pub-dates><date><style  face="normal" font="default" size="100%">DEC</style></date></pub-dates></dates><number><style face="normal" font="default" size="100%">8</style></number><publisher><style face="normal" font="default" size="100%">SAGE PUBLICATIONS INC</style></publisher><pub-location><style face="normal" font="default" size="100%">2455 TELLER RD, THOUSAND OAKS, CA 91320 USA</style></pub-location><volume><style face="normal" font="default" size="100%">11</style></volume><pages><style face="normal" font="default" size="100%">1035-1042</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p&gt;A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The K., values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH4Cl, 3.25 mM for MgCl2, and 2.5 mM for adenosine triphosphate. The K-m value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25 degrees C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z' factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 mu g/ml concentration. The IC50 values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 mu M and 30 pM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z' and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.&lt;/p&gt;</style></abstract><issue><style face="normal" font="default" size="100%">8</style></issue><work-type><style face="normal" font="default" size="100%">Article</style></work-type><custom3><style face="normal" font="default" size="100%">&lt;p&gt;Foreign&lt;/p&gt;</style></custom3><custom4><style face="normal" font="default" size="100%">2.218</style></custom4></record></records></xml>