Studies of a methotrexate binding protein fraction from L1210 lymphocyte plasma membranes

Abstract

[ 14 C]- N-ethylmaleimide has been used to label binding sites for methotrexate in the plasma membranes of L 1210 cells previously ‘protected’ from reaction with unlabeled N-ethylmaleimide by the presence of methotrexate. A [ 14 C]- N-ethylmaleimide labeled protein of molecular weight 56,000 was subsequently identified by electrophoresis of crude plasma membranes in sodium dodecyl sulfate containing polyacrylamide gels. Sepharose—methotrexate affinity chromatography was used to isolate an active methotrexate binding protein fraction from L 1210 lymphoma cell plasma membranes. This protein fraction was isolated from crude plasma membrane preparations and from a detergent extract of intact cells. The latter method carried out in the presence of 0.01% TritonX- 100 permits the rapid extraction of plasma membrane proteins from intact L 1210 cells in the absence of extensive cellular destruction. The methotrexate binding capacity of the extracted protein material has been demonstrated. The protein fraction purified by affinity chromatography contains 3 major protein components of molecular weights 67,000, 63,000 and 56,000 as determined by sodium dodecyl sulfate gel electrophoresis.