D N Joshi1, J S Patel, S J S Flora, K Kalia. 1. Laboratory of Biochemistry, B.R.D. School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, 388 120, Gujarat, India.
Abstract
OBJECTIVE: The aim of this study is to examine arsenic accumulation by Pseudomonas stutzeri and its response to some thiol chelators, DMPS and MiADMSA. METHODS: Determination of arsenic accumulation by Pseudomonas sp. was carried out using an atomic absorption spectrophotometer, a TEM and an EDAX. Arsenate reductase enzyme assay was carried out from a cell-free extract of Pseudomonas sp. The effect of chelating agents on arsenite accumulation was analyzed. Total cellular proteins were analyzed using 1-D SDS-PAGE. RESULTS: Pseudomonas sp. exhibited a maximum accumulation of 4 mg As g(-1) (dry weight). TEM and EDAX analysis showed the presence of As-containing electron-dense particles inside the cells. Data on arsenate reductase enzyme kinetics yielded a K (m) of 0.40 mM for arsenate and a V (max) of 5,952 mumol arsenate reduced per minute per milligram of protein. The chelating agents MiADMSA and DMPS were found to reduce the arsenic accumulation by 60 and 35%, respectively, whereas the presence of both chelating agents in medium containing cells pretreated with arsenite reduced it by up to 90%. The total protein profile of the cellular extract, obtained by 1-D SDS-PAGE, indicated five upregulated proteins, and three of these proteins exhibited differential expression when the cells were grown with MiADMSA and DMPS. CONCLUSION: This study shows a new approach towards arsenic detoxification. A combination treatment with MiADMSA and DMPS may be useful for removing intracellular arsenic. The proteins that were found to be induced in this study may play an important role in the extrusion of arsenic from the cells, and this requires further characterization.
OBJECTIVE: The aim of this study is to examine arsenic accumulation by Pseudomonas stutzeri and its response to some thiol chelators, DMPS and MiADMSA. METHODS: Determination of arsenic accumulation by Pseudomonas sp. was carried out using an atomic absorption spectrophotometer, a TEM and an EDAX. Arsenate reductase enzyme assay was carried out from a cell-free extract of Pseudomonas sp. The effect of chelating agents on arsenite accumulation was analyzed. Total cellular proteins were analyzed using 1-D SDS-PAGE. RESULTS:Pseudomonas sp. exhibited a maximum accumulation of 4 mg As g(-1) (dry weight). TEM and EDAX analysis showed the presence of As-containing electron-dense particles inside the cells. Data on arsenate reductase enzyme kinetics yielded a K (m) of 0.40 mM for arsenate and a V (max) of 5,952 mumol arsenate reduced per minute per milligram of protein. The chelating agents MiADMSA and DMPS were found to reduce the arsenic accumulation by 60 and 35%, respectively, whereas the presence of both chelating agents in medium containing cells pretreated with arsenite reduced it by up to 90%. The total protein profile of the cellular extract, obtained by 1-D SDS-PAGE, indicated five upregulated proteins, and three of these proteins exhibited differential expression when the cells were grown with MiADMSA and DMPS. CONCLUSION: This study shows a new approach towards arsenic detoxification. A combination treatment with MiADMSA and DMPS may be useful for removing intracellular arsenic. The proteins that were found to be induced in this study may play an important role in the extrusion of arsenic from the cells, and this requires further characterization.
Authors: Zijuan Liu; Jian Shen; Jennifer M Carbrey; Rita Mukhopadhyay; Peter Agre; Barry P Rosen Journal: Proc Natl Acad Sci U S A Date: 2002-04-23 Impact factor: 11.205