MEMBRANE-TRANSPORT BY MURINE LYMPHOCYTES .2. APPEARANCE OF THYMIDINE TRANSPORT IN CELLS FROM CONCANAVALIN A-STIMULATED MICE

  • 1 January 1977
    • journal article
    • research article
    • Vol. 118  (4) , 1328-1334
Abstract
Membrane transport of thymidine and adenosine was investigated in bulk murine nonadherent spleen cells from animals treated with the lectin concanavalin A. Separation of cells from radioactive medium was achieved by means of the oil microfuge technique which permits incubation periods as short as 4 s.c. It was possible to distinguish between the membrane-linked function of transport and uptake which includes subsequent accumulation and metabolism of the transport solute. It was previously reported that cells derived from untreated mice do not translocate thymidine in a carrier-mediated fashion. In this report, cells from concanavalin A-treated mice showed 2 membrane transport systems for thymidine, a high affinity system (Km = 160 .mu.M) and one with low affinity (Km = 4 mM). The transport of thymidine conformed to the usual criteria for membrane carrier-linked function: increased uptake with time, substrate saturability and chenical specificity. The transport of adenosine by cells from lectin-treated mice was similar to cells from untreated animals. A specific membrane-linked activity is altered differentially in the course of mitogen-effected lymphocyte stimulation. Determination of DNA synthesis by the standard method gave values which were as much as 700% too high, since the counts obtained by direct precipitation with TCA [trichloroacetic acid] of cells incubated with radiolabeled thymidine exceed the cell-associated radiolabel obtained by the rapid sampling technique. With lectin-stimulated cells, the discrepancy observed was up to 3 times greater. The validity of the standard assay for blastogenesis must be viewed with caution.