naa a22 4500 510746535 CHVBK 20180411083048.0 cr unu---uuuuu 180411e20130301xx s 000 0 eng 10.1007/s12192-012-0376-9 doi (NATIONALLICENCE)springer-10.1007/s12192-012-0376-9 Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells [Elektronische Daten] [Tuoen Liu, Allison Dean, Saint Ashwini, Peter Sheridan, Alok Bhushan, James Lai, Shousong Cao, Christopher Daniels] We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein. Cell Stress Society International, 2012 Leukemia nationallicence HSC70 nationallicence Drug resistance nationallicence Methotrexate nationallicence Methotrexate-binding protein nationallicence Liu Tuoen Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA aut Dean Allison Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT, USA aut Ashwini Saint Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut Sheridan Peter Department of Biological Sciences, Idaho State University, Pocatello, ID, USA aut Bhushan Alok Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut Lai James Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut Cao Shousong Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, 14263, Buffalo, NY, USA aut Daniels Christopher Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut Cell Stress and Chaperones Springer Netherlands 18/2(2013-03-01), 223-234 1355-8145 18:2<223 2013 18 12192 https://doi.org/10.1007/s12192-012-0376-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12192-012-0376-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Liu Tuoen Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA aut NATIONALLICENCE 700 1- Dean Allison Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT, USA aut NATIONALLICENCE 700 1- Ashwini Saint Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut NATIONALLICENCE 700 1- Sheridan Peter Department of Biological Sciences, Idaho State University, Pocatello, ID, USA aut NATIONALLICENCE 700 1- Bhushan Alok Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut NATIONALLICENCE 700 1- Lai James Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut NATIONALLICENCE 700 1- Cao Shousong Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, 14263, Buffalo, NY, USA aut NATIONALLICENCE 700 1- Daniels Christopher Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, 970 South 5th Avenue, Campus Box 8334, 83209, Pocatello, ID, USA aut NATIONALLICENCE 773 0- Cell Stress and Chaperones Springer Netherlands 18/2(2013-03-01), 223-234 1355-8145 18:2<223 2013 18 12192 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer