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FIG. 2. Northern analysis of MnSOD mRNA in transgenic mice. Poly(A)+ RNA from the skin of control (—) and transgenic (+) mice was extracted on a 1.1% (v/v) formaldehyde-agarose gel, transferred to nitrocellulose, and probed with 32P-labeled human MnSOD cDNA.

gels, an SOD activity gel assay is performed according to the method described by Beauchamp and Fridovich.24 Tissue homogenate is prepared in 50 mM potassium phosphate buffer, pH 7.8. Two hundred micrograms of protein per lane is elec-trophoresed through a nondissociating riboflavin-polyacrylamide gel consisting of a 5% (w/v) stacking gel, pH 6.8, and a 10% (w/v) running gel, pH 8.8, at 4°. To visualize SOD activity, gels are first incubated in 2.43 mM nitroblue tetrazolium in deionized water for 15 min and then in 0.028 mMriboflavin and 280 mM N,N,N\N'~ tetramethyl ethylenediamine in 50 mM potassium phosphate buffer, pH 7.8, for 15 min in the dark. Gels are then washed with deionized water and illuminated under fluorescent light until clear zones of SOD activity are distinctly evident. The activity gel assay, which facilitates the identification of SOD isoenzymes, demonstrates the presence of human MnSOD activity, and is the only method that permits distinction between MnSOD activity derived from the transgene and endogenous mouse MnSOD.

Immunogold Staining of MnSOD

Tissues are cut into 1-mm3 blocks, fixed in Carson's modified Millonig fixative [4% (v/v) formaldehyde in 0.16Msodium phosphate buffer, pH 7.2], and processed

24 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971).

for immunogold electron microscope localization of MnSOD in the mitochondria as previously described.9 With the exception of the antibody used, the procedures are identical to those described in detail for the detection of 4-hydroxy-2-nonenal byT. D. Oberley et a/.24a

Assessment of Antioxidant Status

In addition to MnSOD, the activities of Cu,ZnSOD, GSH peroxidases, and catalase, and levels of important low molecular weight antioxidants, i.e., ascorbic acid, GSH, and vitamin E (a-tocopherol), are measured to determine whether altered expression of MnSOD results in compensatory or adaptive changes in antioxidant status.

Immediately after sacrifice, tissues are removed, trimmed, and homogenized with 1.15% (w/v) KC1 in 0.05 M phosphate buffer, pH 7.4. Portions of the ho-mogenate, after centrifugation at 400g for 5 min to remove cell debris, are used to measure SOD activity by monitoring the inhibition of nitroblue tetrazonium-bathocuproline sulfonate reduction, according to the method of Spitz and Oberley.25 The activity of MnSOD is assayed in the presence of 5 mM NaCN, which inhibits Cu,ZnSOD, at 560 nm in 0.05 M potassium phosphate buffer, pH 7.8. NaCN is incubated with the reaction mixture for 30 min before adding xanthine oxidase. In the absence of NaCN, total SOD activity is measured, and the activity of Cu,ZnSOD is calculated. The activity of SOD is expressed as units per minute per milligram protein, and 1 unit is defined as the amount of enzyme required to inhibit nitroblue tetrazonium reduction at 50% maximum activity. Catalase activity in 9000g supernatant is measured spectrophotometrically at 240 nm, using hydrogen peroxide as substrate in 0.05 M potassium phosphate buffer, pH 7.O.26 The activity of catalase is expressed as micromoles of hydrogen peroxide reduced per minute per milligram protein. The activities of GSH peroxidases in 9000g supernatant are measured by monitoring NADPH oxidation at 340 nm, using hydrogen peroxide or cumene hydroperoxide as substrate in 0.05 M Tris-HCl, pH 7.6.27 The use of cumene hydroperoxide as substrate allows for measuring total GSH peroxidase, whereas Se-GSH peroxidase activity is measured when hydrogen peroxide is used as substrate. The activity of the enzyme is expressed as nanomoles of NADPH oxidized per minute per milligram protein. The activities of MnSOD, Cu.ZnSOD, catalase, and Se-GSH peroxidase in the heart and skeletal muscle of MnSOD transgenic and control mice are shown in Fig. 3.

Measurements of GSH peroxidases and catalase are relatively simple and straightforward, and are not normally subject to methodological errors. On the

24a T. D. Oberley, S. Toyokuni, and L. I. Szweda, Free Rad. Biol. Med. 27, 695 (1999).

25 D. R. Spitz and L. W. Oberley, Anal. Biochem. 195, 133 (1989).

26 R. F. Beers and J. W. Sizer, J. Biol. Chem. 195, 133 (1952).

27 R. A. Lawrence and R. F. Burk, Biochem. Biophys. Res. Commun. 71, 952 (1976).

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Mn-SOD CuZn-SOD Catalase Se-GSH Px

Fig. 3. Activities of antioxidant enzymes in the heart and skeletal muscle of 12-week-old male MnSOD transgenic (T) and control (C) mice. The activity of SOD is expressed as units per minute per milligram protein, that of catalase as micromoles per minute per milligram protein, and that of Se-GSH peroxidase (Px) as nanomoles per minute per milligram protein. The data are shown as means ± standard deviation (n = 6). An asterisk denotes a significant difference (p < 0.01) between the mean values of transgenic and control mice.

Mn-SOD CuZn-SOD Catalase Se-GSH Px

Fig. 3. Activities of antioxidant enzymes in the heart and skeletal muscle of 12-week-old male MnSOD transgenic (T) and control (C) mice. The activity of SOD is expressed as units per minute per milligram protein, that of catalase as micromoles per minute per milligram protein, and that of Se-GSH peroxidase (Px) as nanomoles per minute per milligram protein. The data are shown as means ± standard deviation (n = 6). An asterisk denotes a significant difference (p < 0.01) between the mean values of transgenic and control mice.

other hand, methodological inaccuracies in SOD measurements can easily result if optimal conditions for inhibiting Cu,ZnSOD activity by NaCN as well as the proper amount of enzyme preparation to be used are not established on a tissue-by-tissue basis before measurement.25

Freshly prepared tissue homogenate is used to measure low molecular weight antioxidants ascorbic acid, GSH, and vitamin E (a-tocopherol). After acid precipitation of protein, the level of ascorbic acid in the supernatant is measured after reaction with 2,4-dinitrophenylhydrazine at 515 nm,28 and that of GSH is measured after reaction with 5,5'-dithiobis(2-nitrobenzoic acid) at412 nm.29 After extraction of lipids with hexane, the levels of a-tocopherol are measured by high-performance liquid chromatography (HPLC), using a C18 reversed phase column with fluorescence detection.30 The spectrophotometric measurement of GSH, ascorbic acid, and «-tocopherol is not as specific as the HPLC procedure. However, it allows

28 S. T. Omaye, J. D. Turnbull, and H. E. Sauberich, Methods Enzymol. 62, 3 (1979).

29 J. Sedlack and R. H. Lindsay, Anal. Biochem. 25, 192 (1968).

30 L. J. Hatam and H. J. Kayden, J. Lipid Res. 20, 639 (1979).

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