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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, 1995); 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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