Fig. 3

Permeabilisation with alamethicin can be monitored as NADP-dependent ICDH activity. a Online spectrophotometric data obtained using N. tabacum cell cultures. Numbers below the slopes indicate NADP-ICDH activity rates (nmol min^− 1 g^− 1 (fresh weight)). Subscripts indicate alamethicin (Alam) concentration (μg ml^− 1). Triton X-100 (TX-100) was added to 0.1% (w/v) and EDTA to 25 mM. The assay shows representative activity patterns among 2–7 separate measurements of each kind. Absorbance difference between horizontal lines, 0.2 A. b The effect of alamethicin on the ICDH activity in N. tabacum cell cultures (20 mg ml^− 1). The reaction was stopped after 10 min using excess EDTA and measured using a plate reader. Maximum is activity in the presence of 0.1% (w/v) Triton X-100. Bars show SE for n = 3. c ICDH-activity after alamethicin permeabilisation in cells (20 mg ml^− 1) preincubated with 0.2% (w/v) cellulase (+Ce) or boiled cellulase (+BCe). Activity was measured as in (b) using 12 μg ml^− 1 alamethicin. Bars show SE for n = 3. d ICDH activity using 40 μg ml^− 1 alamethicin, in different numbers of A. thaliana seedlings that had been grown for 5 days in H₂O. Reaction was stopped after 1 h with excess EDTA. e ICDH activities in seedlings, as in (d), with different concentrations of alamethicin. Bars show SE for n = 4