首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Irreversible inhibitors of methotrexate transport in L1210 cells: Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate
Authors:Gary B Henderson  Barbara Montague-Wilkie
Institution:Division of Biochemistry, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037 U.S.A.
Abstract:Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37°C, inhibition was rapid (t12 < 1 min), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4°C. Inactivation in this case was qualitatively similar to the corresponding process at 37°C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4°C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.
Keywords:Methotrexate transport  N-Hydroxysuccinimide ester  Affinity labeling  Transport protein  Transport inhibition  Binding site  EDC  1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  Hepes  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
本文献已被 ScienceDirect 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号