Browsing by Subject "Adenosine triphosphatase"
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Item Investigation of the mechanistic basis for the role of Rad50 in double-strand break repair(2006) Bhaskara, Venugopal; Paull, Tanya T.Members of the Rad52 epistasis group, which includes a heterotrimeric complex, formed by Mre11, Rad50 and Nbs1 (Xrs2 in yeast) helps in the protection of cell’s genetic content from DNA doublestrand breaks. The Rad50 component of the human Mre11/Rad50/Nbs1 (Xrs2 in yeast) complex (MRN(X)) belongs to the ABC superfamily of ATPases and is conserved among all organisms and contains Walker A (N-Terminus) and Walker B (C-Terminus) ATPase domains connected by a long coiled-coil region. We show for the first time that Rad50 shows adenylate kinase activity (ATP + AMP ↔ 2ADP) and that this activity is important for tethering of DNA ends. We further show that Rad50 can catalyze “reverse” adenylate kinase activity (2ADP → ATP + AMP) and this activity is stimulated in the presence of linear DNA ends. We also show that the signature motif of Rad50 is essential for all ATP-dependent activities in vivo and in vitro.Item Proton ATPase are involved in metastasis in human breast cancer cell(Texas Tech University, 2001-12) Luo, DefengCancer cells live in a more acidic environment than the normal tissue, but the intracellular pH (pHin) of tumors is more alkaline than in non-tumor cells. This indicates that cancer cells maintain a larger transmembrane pH gradient than normal cells. Our previous studies suggested that vacuolar type H'*^-ATPases (V-H'*'ATPases) that normally reside in acidic organelles may be also expressed at the plasma membrane (pmVATPase) of highly metastatic human tumors. We hypothesize that pmV-ATPase plays a role in maintaining an alkaline intracellular environment favorable for growth, while maintaining an acidic extracellular environment favorable for invasion. Human breast cancer cell lines with distinct metastatic potential (MCF-7, MB-231, MB468 and MB- 435s) were used as experimental models. Immunocytochemical experiments showed that pmV-ATPases are located at the leading edge of metastatic human breast cancer cells. We employed spectral imaging and line scanning confocal microscopy (LSCM) to monitor the pHfn of discrete cellular regions, as well as fluorescence spectroscopy to determine proton fluxes in these cancer cell lines. These approaches are complementary in that they offer unsurpassed spatial, temporal and spectral resolution. Our data show that the pHin is more alkaline at the leading than at the lagging edge in the more invasive cells. The magnitude ofthe pHin gradient is larger in the highly than in the lowly metastatic cells. The proton fluxes are faster in the highly than in the lowly metastatic cells. Pharmacological and ion substitution experiments indicated that pmV-H"^ATPase expression was more elevated in the highly than in lowly metastatic cells. Invasion assays of these cancer cells lines show that bafilomycin Ai inhibits the invasive behavior. These novel observations suggest that the magnitude ofthe pH gradients is determined by pmVH^ ATPase, and pmV-H^ ATPase is involved in the invasion ofthe cancer cells.