NDLI: Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

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Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

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Cell Stress and Chaperones : Volume 18, Issue 1, January 2013

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Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

Content Provider	Springer Nature Link
Author	Liu, Tuoen Dean, Allison Ashwini, Saint Sheridan, Peter P. Bhushan, Alok Lai, James C. K. Cao, Shousong Daniels, Christopher K.
Copyright Year	2012
Abstract	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 66–68 kDa. 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.
Starting Page	223
Ending Page	234
Page Count	12
File Format	PDF
ISSN	13558145
Journal	Cell Stress and Chaperones
Volume Number	18
Issue Number	2
e-ISSN	14661268
Language	English
Publisher	Springer Netherlands
Publisher Date	2012-10-23
Publisher Place	Dordrecht
Access Restriction	Subscribed
Subject Keyword	Leukemia HSC70 Drug resistance Methotrexate Methotrexate-binding protein Biomedicine general Cell Biology Biochemistry Immunology Cancer Research Neurosciences
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry

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Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.

Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

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Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

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Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells