A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors |
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Authors: | Sandra S Susten Robert J Kempton Angelique M Black James H Freisheim |
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Institution: | 1. Department of Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati, OH 45267, U.S.A.;2. Department of Physical Sciences, Northern Kentucky University, Highland Heights, KY 41076, U.S.A. |
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Abstract: | A dansyl-l-lysine analogue of methotrexate, , is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for 3H]methotrexate transport with a Ki of 7.02 μM, a value virtually identical to the Kt for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase. |
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Keywords: | DHFR dihydrofolate reductase (EC 1 5 1 3) MTX methotrexate 4-amino-4-deoxy-10-methylpteroylglutamic acid MTX-F fluorescein-diaminopentane-methotrexate ALD ALF To whom correspondence should be addressed |
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