Fig. 3. Size-exclusion limit for peroxisomal membrane permeability. (A) Effect of PEGs on solubilization of enzymes from peroxisomes during rat liver homogenization. Livers were perfused to wash out erythrocytes and samples (2 g) were homogenized in 10 ml of isolation-medium 2 containing 0.1 M PEGs of different molecular masses (left panel) or PEG 1500 at different concentrations (right panel). The homogenates were centrifuged at 100,000 g_max for 60 minutes. Activities of catalase (), L-hydroxyacid oxidase () and protein content () were determined in the whole homogenate (total activity) and in the supernatant (unsedimentable activity). Columns show the unsedimentable activity of catalase (1) and L- hydroxyacid oxidase (2) after homogenization of liver samples in isolation-medium 1. Values are means±s.d. (n=3-4). (B) Illustration explaining bottle-stopper experiment. Grey circles, PEGs with different molecular size; black circles, uric acid molecules. (1) PEG molecules that are much smaller than the pore of the membrane channel can therefore freely diffuse with uric acid into peroxisomes; (2) PEG molecules of a size similar to the pore of the channel. PEGs penetrate into the channel very slowly and prevent rapid diffusion of uric acid through the channel; (3) PEG molecules that are too large to penetrate into the channel. Molecules of uric acid easily move through the channel into peroxisomes. (C) Free activity of urate oxidase () in purified peroxisomal fractions in the presence of PEGs of different molecular sizes. The incubation medium for determination of urate oxidase activity contained 0.16 M PEGs. Values given are means±s.d. (n=3-4). (D) Optical density tracings in swelling experiments with purified peroxisomes incubated at 25°C in the presence of PEGs: (1) control, addition of the buffer only; (2) PEG 200; (3) PEG 400; (4) PEG 600; (5) PEG 1000; (6) PEG 1500. 200 µl of PEG solution (40%, w/v) was added to 600 µl of peroxisomes suspended in isolation-medium 2 (OD₅₂₀=0.5). The delay in recording, owing to the interruption caused by adding PEGs, was 1 minute. (E) Dependence of OD₅₂₀ value on the molecular size of PEGs. Data were collected 1 minute () and 10 minutes () after mixing the peroxisomes with PEGs. Results are shown as the difference in OD (OD) of the samples containing PEG relative to the control without solute (zero level). Data of one representative experiment are shown. Experiments with PEGs (C-E) were performed with peroxisomal preparations isolated by the standard procedure.