AMINO ACID COMPOSITION OF STEM BROMELAIN.
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Cells of the original line of lymphoma 6C3HED, which regularly prove susceptible to the effects of guinea pig serum in vivo, were cultured in Eagle's medium devoid of L-asparagine; after a latent period of 2 or more weeks, during which time the cell population declined markedly, some of the cells began to proliferate, and thereafter continued vigorous growth. On implantation into mice the proliferating cells were found, however, to have completely and permanently lost their susceptibility to the effects of guinea pig serum. By contrast, when cultures of the original line of 6C3HED cells were prepared in Eagle's medium to which L-asparagine was added in a concentration of 20.0 mg/liter or more, they proliferated vigorously from the beginning; after long periods of growth in the enriched medium in vitro they remained susceptible to the effects of guinea pig serum upon test in vivo. Other amino acids, purines, and pyrimidines were unable to substitute for L-asparagine in this relation. Furthermore, a variant subline of 6C3HED cells which had become insensitive to guinea pig serum under in vivo conditions did not require L-asparagine for growth in tissue culture. It seems plain from the findings as a whole, that in 6C3HED cells, L-asparagine dependence in vitro is associated with the in vivo character of guinea pig serum sensitivity, and conversely L-asparagine independent variants are insusceptible to the effects of guinea pig serum. The implications of the findings complement those of a companion paper in which direct evidence is provided that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects.
A number of the properties of the L-asparaginase present in guinea pig serum have been examined and shown to be indistinguishable from those of the agent responsible for inhibiting cells of lymphoma 6C3HED in vivo. The patterns of instability of the enzyme to changes in temperature and pH were found to parallel closely those of the antilymphoma agent. L-Asparaginase activity was essentially absent from the serum of newborn guinea pigs and this failed to inhibit 6C3HED cells. On separating guinea pig serum proteins by salt precipitation, electrophoresis, and chromatography on DEAE cellulose, antilymphoma activity was found only in fractions which contained L-asparaginase.