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Rensselaer, NY, United States

Oliver J.C.,Purdue University | Linger R.S.,University of Charleston | Chittur S.V.,Purdue University | Chittur S.V.,Center for Functional Genomics | Davisson V.J.,Purdue University
Biochemistry | Year: 2013

Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5′-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5′-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites. © 2013 American Chemical Society. Source


Grant
Agency: Cordis | Branch: FP7 | Program: CSA-SA | Phase: REGPOT-2007-1-01 | Award Amount: 535.10K | Year: 2008

Over the past decade significant resources have been committed to development of capacities for genomic research in Croatia. These efforts have lead to formation of the Centre for Functional Genomics of the Faculty of Medicine and University Hospital, University of Zagreb. Within the national confines, Centre for Functional Genomics formed strong links with other Croatian groups interested in genomic research, as well as with recognised centres of excellence in European Union (in Scotland, Portugal and Germany), and also with the Harvard University (USA). Initially formed through common research interests, these collaborations resulted in award of several research grants from the Croatian Ministry of Science, as well as international grants with EU partners (from The British Council, Royal Society UK, The Wellcome Trust, Medical Research Council UK, National Institutes of Health USA and European Union). This proposal therefore seeks to strengthen and reinforce Centre for Functional Genomics of the Zagreb Medical School and its existing links with Croatian and EU centres. The timing in Croatia is now very favourable for building on recent successes that include employment of promising researchers in the field of genomics, securing substantial competitive international research funding and publication of research results in high-impact journals. Support from the European Commission to this initiative would secure further development of genomic advances in the Croatian biomedical scientific community, integration of existing capacities within the country and bolstering the links with international centres of excellence to ensure the sustainability of existing programmes. These activities could also have potential impact on local economy, social environment and health. This would result in creation of a highly competitive group of scientists from the EU convergence regions that could get involved in further applications within the EU FP7 program.


Jain R.,University at Albany | Doyle F.,University at Albany | George A.D.,University at Albany | Kuentzel M.,Center for Functional Genomics | And 3 more authors.
Methods in Molecular Biology | Year: 2010

Microarrays are extensively used to evaluate the effects of compounds on gene expression in the cells. Most of the studies so far have analyzed the transcriptome of the cell. The basic assumption of this approach is that the changes in gene expression occur at the level of transcription of a gene. However, changes often occur at the posttranscriptional level and are not reflected in the analysis of whole transcriptome. We have pioneered the development of "ribonomic profiling" as a high-throughput method to study posttranscriptional regulation of gene expression in the cell. This method is also often referred to as RIP-CHIP. In this chapter, we describe how to use the RIP-CHIP technology to assess the posttranscriptional changes occurring in the cell in response to treatment with a drug. © 2010 Humana Press, a part of Springer Science+Business Media, LLC. Source

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