ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 229, No. 2, March, pp. 544-554, 1984 The Use of Radioactive Cysteine Methyl Ester for Labeling Glycosylated Molecules Oxidized by Periodate or Neuraminidase plus Galactose Oxidase RICHARD N. MITCHELL,’ EARL H. HARRISON: AND WILLIAM E. BOWERS3 The Rockefeller University, New York, New York 10021 Received September 7, 1983, and in revised form November 10, 1983 Treatment of rat lymph node cells with periodate or neuraminidase plus galactose oxidase initiates blast transformation and cell division of T lymphocytes. Either treat- ment introduces aldehyde functions onto glycosylated molecules of the plasma mem- brane. 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 lo-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 incor- porated 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 re- agents labeled with isotopes other than tritium, including those like 35S, which are much stronger radioactive emitters. Treatment of intact cells with periodate, functions on glycosylated molecules of the or with the enzymes neuraminidase plus plasma membrane. Mild periodate oxida- galactose oxidase, introduces aldehyde tion leads to the generation of a C-7 or C- 8 aldehyde derivative of terminal sialic acid 1 Present address: Harvard Medical School, Boston, residues of glycoproteins and glycolipids Mass. 02115. (1, 2), whereas the enzymatic treatment *To whom correspondence should be addressed: introduces an aldehyde function at the C- Department of Biological Chemistry, Wright State 6 position of initially penultimate galactose University, School of Medicine, Dayton, Ohio 45435. or N-acetylgalactosamine residues (3). ’ Present address: The Mary Imogene Bassett Hos- Subsequent reduction of these aldehydes pital, Cooperstown, N. Y. 13226. with high-specific-activity rH]borohydride 0003-9861/84 $3.00 544 Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.