Abstract
The proteins of [35S]methionine-labeled membranes of six lymphoid tumors which expressed DBA/2 genes were examined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels in order to identify molecular differences among the tumors. The tumors were labeled with [35S]methionine, their membranes were isolated, and sodium dodecyl sulfate-polyacrylamide gradient gels of the reduced and alkylated membrane proteins were electrophoresed. In autoradiograms of these gels, 110 protein bands were identified. The pattern of membrane proteins was complex, showing several bands which varied in their presence or quantity among the tumors. At least 20 proteins were exposed to the cell surface, as judged by susceptibility to lactoperoxidase-mediated radioiodination. The correspondence of some gel bands to serologically recognized membrane antigens was established by electrophoresis of radiolabeled H-2K, H-2D, and Lyb-4.1 antigens and β2-microglobulin from double antibody precipitates in parallel to membrane samples. Lyb-4.1 alloantigen was identified to have a molecular weight of 44,000 and did not bind β2-microglobulin.
In addition, this study defined restrictions on this technique arising from the use of [35S]methionine, which was synthesized in Escherichia coli which were then acid hydrolyzed. The neutralized hydrolysate was used without chromatographic purification as an inexpensive source of [35S]methionine. The high salt concentration of this solution restricted conditions for incorporation of [35S]methionine into lymphoblasts. Optimal incorporation conditions were defined with respect to the duration of incubation, the volume and pH of the incubation mixture, and the amount of methionine. The advantage of this technique lay in the use of large quantities of inexpensive [35S]methionine for multiple examinations of different cell lines or in relatively large preparations of radiolabeled membrane proteins for chemical studies.
This work was supported by Basil O'Connor Starter Research Grant 5114 from the National Foundation-March of Dimes; by a grant from the American Cancer Society, Massachusetts Division, Inc., by Research Contract NO1-CB-64072 from the National Cancer Institute; and by the United States Naval Research and Development Command Work Unit ZF 51.524.013.1029.
The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The animals used in this study were handled in accordance with the provisions set forth in the current edition of the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.
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