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First published online October 27, 2004
doi: 10.1242/10.1242/jcs.01485

Journal of Cell Science 117, 5633-5642 (2004)
Published by The Company of Biologists 2004
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The rat liver peroxisomal membrane forms a permeability barrier for cofactors but not for small metabolites in vitro

Vasily D. Antonenkov1,*, Raija T. Sormunen2 and J. Kalervo Hiltunen1,*

1 Department of Biochemistry and Biocenter Oulu
2 Department of Pathology, University of Oulu, PO Box 3000, FI-90014 Oulu, Finland



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  		Fig. 1. Purity of rat liver peroxisomal fraction. Electron micrograph of purified peroxisomes from the Nycodenz gradient. Scale bar, 2 µm.

 


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  		Fig. 2. Latency of peroxisomal enzymes. (A) Activity of xanthine-oxidizing enzyme in a purified peroxisomal fraction. Values given are means±s.d. (n=4-5). (B) Effect of phospholipase C on the free activity of lactate dehydrogenase. Aliquots of purified peroxisomes in 20 mM MOPS, pH 7.4 (0.4 mg protein/ml) were incubated at 37°C with 3 mM CaCl2 ({blacktriangleup}), 1 U/ml phospholipase C ({bullet}), CaCl2 with phospholipase C ({diamondsuit}) or without any additions ({blacksquare}). The reaction was interrupted by 10 mM EDTA (final concentration). Panels B-D show the free activity relative to 100% of the total activity in the same sample. (C) Thermal treatment of purified peroxisomes. Aliquots of peroxisomes in isolation-medium 2 (0.4 mg protein/ml) were incubated for 3 minutes at different temperatures and then diluted with 5 volumes of the same ice-cold medium. The free activities of catalase ({blacktriangleup}), urate oxidase ({diamondsuit}) and L-{alpha}hydroxyacid oxidase ({blacksquare}) were immediately determined. The total activity of the oxidases after thermal treatment of peroxisomal suspension (relative to activity without treatment) at 37°C, 45°C, 50°C, 55°C and 60°C were: 100%, 104%, 98%, 66% and 43% for urate oxidase and 97%, 98%, 94%, 86% and 52% for L-{alpha}hydroxyacid oxidase, respectively. Notice the appearance of the latency of urate oxidase and L-{alpha}hydroxyacid oxidase at 50°C while the total activity of the enzymes was not suppressed at this temperature. (D) Proteinase K treatment of purified peroxisomes. Aliquots of the particles in 20 mM MOPS, pH 7.4 (0.4 mg protein/ml) were incubated in the presence of 0.6 U/ml proteinase K at 37°C. The reaction was stopped by the addition of 1 mM PMSF (final concent ration). The left panel shows the activity of the enzymes in the samples incubated with proteinase K and 0.05% (w/v) Triton X-100 (solid line). In the parallel samples the detergent was added after the incubation with proteinase K (hatched line). Data are presented as a percentage of activity relative to the control (without incubation). The right panel displays the free activity of enzymes in the samples incubated with (solid line) or without (hatched line) proteinase K. Catalase,{bullet}; urate oxidase, {blacktriangleup}; L-{alpha}hydroxyacid oxidase, {blacksquare}; lactate dehydrogenase,{diamondsuit}.

 


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  		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 gmax for 60 minutes. Activities of catalase ({blacksquare}), L-{alpha}hydroxyacid oxidase ({diamondsuit}) and protein content ({blacktriangleup}) were determined in the whole homogenate (total activity) and in the supernatant (unsedimentable activity). Columns show the unsedimentable activity of catalase (1) and L-{alpha} 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 ({blacksquare}) 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 ({blacksquare}) and 10 minutes ({bullet}) after mixing the peroxisomes with PEGs. Results are shown as the difference in OD ({Delta}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.

 


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  		Fig. 4. Cofactors slowly penetrate into peroxisomes. (A) The effect of cofactors on the osmotic behavior of purified peroxisomes. The OD520 was recorded at 25°C after mixing a peroxisomal suspension (OD520=0.6) with: (1) NAD+ or (2) NADP+ (each at 25 mM, final concentration). Control sample (3) was mixed with buffer only (see legend Fig. 3D for further details). (B) Incubation of peroxisomes with cofactors. Suspension of peroxisomes (OD520=0.6) in 20 mM MOPS, pH 7.4 containing 1 mM EDTA was saturated 20 minutes at 25°C with 0.2 mM NADPH (final concentration) in a total volume of 0.8 ml. At the end of incubation, 40 µl of enzyme-substrate mixture (50 mM Tris-Cl, pH 8.0; 0.4 mM 2-oxoglutarate; 0.1 mM ADP; 40 mM ammonium acetate and 10 U/ml glutamate dehydrogenase; each at final concentrations) designed for a sudden oxidation of NADPH was added. The absorbance spectrum (320-380 nm) was measured within 1 minute. The reference sample was incubated in the same conditions as described above, except that the enzyme-substrate mixture was added before the saturation with NADPH. Graphs within B are numbered as follows: (1) sample saturated with NADPH; (2) same as 1 after addition of 5 µl Triton X-100 (10%, w/v); (3) peroxisomes in sample cuvette incubated with NADPH after addition of the enzyme-substrate mixture; (4) same as 3 after addition of Triton X-100. (C) Effect of oxamate on free lactate dehydrogenase activity in a purified peroxisomal fraction. Peroxisomes were treated with 2',5'-ADP-Sepharose as described (Materials and Methods) and incubated 5 minutes at 25°C with oxamate at different concentrations. Total and free activities of lactate dehydrogenase were immediately determined. Free activity (C-E) was estimated relative to total activity (100%) in the same sample. The measurements were repeated several times. Data from one representative experiment are shown. (D) Dependence of lactate dehydrogenase free activity on NADH concentration. Lactate dehydrogenase activity was measured at different NADH concentrations and a fixed concentration of pyruvate (1.0 mM, final concentration). Values given are means±s.d. (n=4-5). (E) Dependence of urate oxidase free activity on uric acid concentration. Urate oxidase forms a crystalline structure inside peroxisomes called a nucleoid (Masters and Crane, 1995Go); this explains the absence of leakage of the enzyme from broken particles. Presumably almost all the free activity of urate oxidase arises from the enzyme that is present inside the particles.

 

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