Sialic acid containing glycoprotein fragments were removed from the surface of the TA3 mammary adenocarcinoma ascites cell of the strain A mouse by short-term incubations with TPCK-trypsin at 4". The material eluted in the void volume of a Bio-Gel P-100 column, after fractionation on Bio-Gels P-4 and P-30, represented 9 % of the carbohydrate and protein material removed from the cells but 31 % of the total surface sialic acid. It was composed of approximately 70 x carbohydrate and 30 % protein and contained four carbo-I t was recently suggested (Sanford and Codington, 1971) that the reduced transplantability of neuraminidase-treated TA3 mammary adenocarcinoma ascites cells of the strain A mouse in the foreign strain C3H mouse (Sanford, 1967) may be due to a serum factor which was demonstrated to be toxic in r;itro to the cells after neuraminidase treatment. In a previous investigation of the sialic acid containing glycoproteins at the surface of this cell, Codington et al. (1970) demonstrated that peptide and carbohydrate material removed from the cells by chelating agents or proteolytic enzymes had widely different compositions, and all samples exhibited marked heterogeneity .The mixture of glycoprotein fragments initially cleaved from the cells by mild treatment with TPCK-trypsin at 15-22" contained the highest proportion of sialic acid, suggesting that the glycoprotein structures richest in sialic acid were probably located at the periphery of the TA3 cell. This paper describes the fractionation by gel filtration of material obtained by short-term successive incubations with TPCKt From the laboratory for Carbohydrate Research,

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A variety of differences between normal and leukemic cells have been reported, involving such diverse characteristics as alkaline phosphatase content,"' 12 glycolytic rate, and nucleic acid metabolism.6 In most cases, however, the differences observed could have been due to variations in cell maturation levels rather than to leukemia-specific changes. We still do not know whether leukemia results from a malignant change within the leucocyte population or from an increase in essentially normal cells due to interference with leucocyte control mechanisms.In our laboratory, we have been investigating an agglutinin present in certain wheat germ lipase preparations which produces more clumping in murine tumor cells than in isologous normal cells.3 Hakamori and Jeanloz have found that the cell surface material involved in the reaction is also present in a glycolipid isolated from a human adenocarcinoma.7' 8 The lipase enzyme itself is not responsible for the agglutination observed since enzyme inactivation by heat or by p-chloromercuribenzoic acid does not destroy agglutinating activity. We have shown that the increased reactivity of tumor cells does not merely reflect a cell surface property related to rapid cell growth and division since spleen cells from newborn animals do not clump more than those from adults, nor do cells from regenerating liver clump more than those from normal liver. It was considered interesting, therefore, to determine whether or not a leukemic cell would behave like a tumor cell in respect to clumping when treated with wheat germ agglutinin.Materials and Methods.-Leucocyte preparation: Leucocyte suspensions were prepared according to a modification of Amos' procedure.' Fifty milliliters of normal blood and 5-10 ml of leukemic blood were used for each experiment. Na2EDTA (5%) was added as an anticoagulant (0.5 ml/5 ml blood), and 5% P.V.P. (1.0 ml/5 ml blood) was used to sediment Ithe red blood cells. The supernatant was centrifuged at 100 X g for 10 min, and the packed cels were resuspended in 0.1 ml of supernatant plasma. The majority of the remaining red blood cells settled out within 1 hr, and the leucocyte-containing supernatant was drawn off to be counted and diluted with calcium-free phosphate-buffered saline (PBS) to a final concentration of 5 X 106 cells/ml. At least 95% of the leucocytes prepared by this method appeared structually intact when tested for trypan blue uptake. (N.B.: Skoog and Beck's fibrinogen citrate method was used for the first two experiments in this series.10) Twenty-four fresh blood samples from 17 patients with leukemia were provided-by Massachusetts General Hospital clinics or private physicians, and most experiments were run in duplicate. Each patient was compared with a normal donor of the same ABO and Rh blood types, and comparisons were made between normal and leukemic of each pair, rather than between experiments.Lymphocyte preparation: Virtually pure lymphocyte preparations were made with a nylon filter. A unit of blood was drawn into a heparin receiving p...

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