Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Abstract

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant ( K i) of approx. 5·10 −7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10 −5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant ( K d) of approx. 1·10 -6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10 6 molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K d of 4–6·10 −7 M, represents approx. 60% of the total saturable binding (14·10 6 molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K d of 2–6 · 10 −8 M, represents less than 10% of the total sites (2 · 10 6 molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.