The use of radioactive cysteine methyl ester for labeling glycosylated molecules oxidized by periodate or neuraminidase plus galactose oxidase.
Treatment of rat lymph node cells with periodate or neuraminidase plus galactose oxidase initiates blast transformation and cell division of T lymphocytes. Either treatment introduces aldehyde functions onto glycosylated molecules of the plasma membrane. Reduction of the aldehydes with borohydride leads to a concentration-dependent inhibition of the mitogenic response. Cysteine methyl ester (Cys(Me], which can form a stable thiazolidine adduct with aldehydes, also inhibits mitogenesis in a concentration-dependent manner. Maximum inhibition is achieved at Cys(Me) concentrations about 10-fold lower than those required for borohydride (0.4 and 5 mM, respectively). [35S]Cys(Me) has been synthesized and compared with [3H]borohydride as a labeling reagent for molecules on the plasma membrane oxidized by periodate or neuraminidase plus galactose oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled whole cell lysates or of crude membrane fractions prepared from labeled cells revealed that the same oxidized molecules are specifically labeled with both reagents. Homogenates of cells labeled with either radioactive reagent were fractionated by differential and isopycnic centrifugation. The fractions were analyzed for radioactivity and for a number of marker constituents localized in various subcellular organelles. Following treatment with either reagent, the radioactivity that was covalently incorporated into macromolecules was primarily associated with sedimentable components that distributed among the fractions like plasma membrane markers. When compared with [3H]borohydride, Cys(Me) offers several advantages as a surface labeling reagent for glycosylated plasma membrane molecules, chiefly the possibility of preparing reagents labeled with isotopes other than tritium, including those like 35S, which are much stronger radioactive emitters.
D000818:Animals; D001894:Borohydrides; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives*; D004261:DNA Replication; D005692:Galactose Oxidase / Q000378:metabolism*; D006027:Glycosides / Q000032:analysis*; D008198:Lymph Nodes / Q000276:immunology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology*; D008297:Male; D009439:Neuraminidase / Q000378:metabolism*; D010504:Periodic Acid; D011865:Radioisotope Dilution Technique; D051381:Rats; D011917:Rats, Inbred Lew; D013462:Sulfur Radioisotopes; D014316:Tritium
D001894:Borohydrides; D006027:Glycosides; D013462:Sulfur Radioisotopes; D014316:Tritium; D010504:Periodic Acid; C009288:metaperiodate; D005692:Galactose Oxidase; D009439:Neuraminidase; D003545:Cysteine; C017507:mecysteine