Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of L1210 cells by treatment with an N-hydroxysuccinimide ester of [3H]methotrexate.
An N-hydroxysuccinimide ester of [3H]methotrexate has been employed to covalently label a specific binding protein that resides in the plasma membrane of L1210 cells. Incorporation of radioactivity into this protein accounted for 55% of total cellular labeling, was half-maximal at a reagent concentration of 27 nM, and was blocked either by prior exposure to unlabeled reagent or by the addition of excess methotrexate. A role for this protein in methotrexate transport was supported by the observations that: (a) similar concentrations of reagent were required for both labeling of the binding protein and irreversible inhibition of transport; (b) the amount of labeled binding protein was comparable to observed levels of transport protein; (c) protection against labeling was afforded by thiamin pyrophosphate, a potent competitive inhibitor of methotrexate transport; and (d) labeling of the binding protein was not observed in a subline of L1210 cells that has a defect in the ability to transport methotrexate. The binding protein could be solubilized from the membrane by various ionic and non-ionic detergents and the covalent bond between the incorporated [3H]methotrexate and the protein was stable to a variety of conditions, including high concentrations of mercaptoethanol and hydroxylamine and extremes of pH. The labeled protein fractionated as a nearly symmetrical peak on Sephacryl S-300 and it appeared as a single band (Mr = 36,000) after electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism* / Q000503:physiopathology; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism*; D051379:Mice; D013763:Tetrahydrofolates / Q000378:metabolism*; D014316:Tritium
D000345:Affinity Labels; D002352:Carrier Proteins; D003902:Detergents; D013763:Tetrahydrofolates; C039702:methotrexate N-hydroxysuccinimide ester; D014316:Tritium; C005984:5-methyltetrahydrofolate; D008727:Methotrexate