DAMI cells possess two nuclear-associated AChE forms that vary in their detergent-vulnerability and stability. Left: AChE activity in intact nuclei of PMA-treated cells is detergent sensitive. Cells were grown for 3 days in culture with or without (C) 10 ng/mL PMA (P) or 930 U/mL TPO (T). Intact nuclei separated from supernatant as detailed in Materials and Methods were homogenized with 0.2%, 0.5%, or 1% Triton (Tr) X-100. AChE activities in these homogenates are presented as average ± standard error in percentages of initial activity measured at 0 time in the presence of 0.1% Triton X-100 (left inset). The data represent the average of three experiments each performed in triplicate assays. Note that following PMA induction, AChE becomes more vulnerable to Triton X-100. Inset: The specific activities (sp. act.) calculated at 0 time in the presence of 0.1% Triton for control, PMA, and TPO cultures are presented as average ± SD hydrolyzed ATCh (10−7 nmol per minute per nucleus). Note that the PMA-treated cells lost a significant fraction (* P < .005) of their enzyme activity. Right: Nuclear-associated AChE in proliferating MK is unstable. Nuclear-associated AChE activities were measured in enzyme preparations from intact nuclei of cells treated 3 days in culture with or without PMA or TPO. The percentage of remaining AChE activities following the noted periods of incubation at 37°C in the presence of 0.1% Triton were calculated as compared with the initial activity at 0 time. The data represent the average of three experiments each performed in triplicate assays. Inset: High magnification of separated intact nuclei stained with DAPI. Size bar = 10 μm.
