PMID: 2119551Jan 1, 1990Paper

Visualization of folate transport proteins by covalent labeling with fluorescein methotrexate

Advances in Enzyme Regulation
J FanF M Huennekens

Abstract

Fluorescein-methotrexate (F-MTX) has been synthesized by an improved procedure and separated via chromatography on DEAE-Trisacryl into the alpha- and gamma-isomers. Purity of each isomer was verified by TLC, HPLC, and absorbance spectra. Identity of the alpha- and gamma-isomers was established by the following biological criteria: the gamma-isomer inhibited dihydrofolate reductase and was hydrolyzed by carboxypeptidase G2 (at the pteroate-glutamate linkage). The alpha-isomer, conversely, was unreactive in both systems, which is consistent with the specificity of these enzymes. Based upon these results, the gamma-isomer was selected for covalent labeling of proteins. Fluorescent bands were observed when the 22 kDa human dihydrofolate reductase and the 18 kDa folate transporter from Lactobacillus casei were treated with gamma-F-MTX (activated by N-hydroxysuccinimide) and subjected to SDS-PAGE. The probe was also useful for visualizing in situ the micromolar folate transport protein in L1210 cells.

References

May 1, 1975·Journal of Medicinal Chemistry·G R GapskiF M Huennekens
Jan 1, 1989·Advances in Enzyme Regulation·L E PopeF M Huennekens
May 22, 1984·Biochemistry·R L Blakley, L Cocco
Jun 1, 1980·Archives of Biochemistry and Biophysics·G B HendersonJ M Whiteley
Mar 1, 1983·Archives of Biochemistry and Biophysics·G B Henderson, E M Zevely

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Citations

Jan 1, 1992·Advances in Enzyme Regulation·J FanF M Huennekens
Aug 12, 2003·American Journal of Physiology. Gastrointestinal and Liver Physiology·Tiesong LiToshiharu Horie

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