Fig. 2. (A) Intracellular accumulation of biotinylated histone proteins (externally added) in cultured Colo-205 cells: quantitative estimation. Prior-neut (prior neutralization): biotinylated histones neutralized with avidin and biocytin before being added to the Colo-205 cells. All subsequent steps including cell permeabilization and estimation of biotinylated histones are as described in Materials and Methods. No detergent: estimation of cell-surface-bound biotin molecules. Biotinylated histones were incubated with Colo-205 cells and following neutralization of surface-bound biotin the remaining biotin was estimated on unlysed cells. Control 37°C and 4°C: the biotinylated histone mixture was incubated with Colo-205 cells at 37°C or at 4°C and after neutralizion with avidin and incubation with biocytin, the cells were treated with detergents. Prior fixation: cells were fixed by formaldehyde prior to the incubation period. Excess histone: as control 37°C but in the presence of excess unlabelled histone (x100 mole/mole). ATP-depleted cells: as control 37°C and ATP depletion was performed as described in Table 1. , nuclei; , cytosol. The amount of biotinylated histone present in the nuclei of cells incubated at 37°C was considered as 100% and was calculated to be 6.2 nmol histone/mg protein, which was estimated to be about 60% of the total added histones. (B) Kinetic studies of histone penetration. Biotinylated histones were incubated with Colo-205 cells at 37°C in the absence () or in the presence () of 0.5 M sucrose and at 4°C (). Pre-incubation with sucrose (0.5 M) was performed as described in Table 1. An optical density (OD) of 0.25 represents 4.7 nmol histone/mg protein. (C) Kinetic studies of histone and importin alpha penetration. Biotinylated histone () and importin alpha () were incubated with Colo-205 cells at 37°C. Penetration of histone and importin was estimated as described in Materials and Methods, except that the incubation was performed in PBS and not in TB as in B.