Actinomycin D binding to isolated deoxyribonucleoprotein and intact cells

Abstract

The binding of actinomycin D (AMD) by isolated deoxyribonucleoprotein (DNP) and chromatin of intact cells has been studied by chemical and autoradiographic experiments with radioactive AMD and by optical titrations. The binding capacity of isolated DNP was found to be one to two orders of magnitude higher than that of DNP in intact cells. In the presence of Ca 2+-ions and at higher ionic strengths the binding capacity of isolated DNP was depressed. EDTA has the opposite effect on intact cells. The degree of AMD binding was highly dependent on the concentration of AMD used. This concentration dependence was most pronounced for intact cells although it was also observed with isolated DNP where two types of binding can be detected. No significant differences were found in the AMD-binding to DNP isolated from hen erythrocytes, human leucocytes and HeLa cells. Differences in AMD-binding capacities were, however, eliminated during the isolation since living hen erythrocytes bound approximately twice as much AMD per unit DNA as HeLa cells which in turn bound 3–4 times as much as human leucocytes. Kinetic studies suggested that there are two phases of binding to the living cells tested: one early rapid phase and a second slower one. The differences in binding capacity were diminished or even reversed if the cells were fixed in ethanol-acetic acid (9:1) prior to incubation with AMD. This fixation also dramatically increased the AMD-binding capacity of all cell types tested. The large proportion of non-AMD-binding cells seen in human lymphocyte populations was found to bind AMD after fixation. The penetration of AMD into living cells was studied with “dry” autoradiography. The results show that the varying extent of binding cannot be explained solely on the basis of differences in penetration. The biological significance of the observations is discussed.