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Fig. 14.1. Recovery of glutathione (GSH). Replicate samples from a stationary cell culture were harvested as described in the Methods. A known amount of pure reduced glutathione was added to subsamples (spike) either to the cell pellet before or after the addition of perchloric acid and all samples analysed in parallel. Amounts of standard pure GSH recovered ranged from 90% to 110%. Values less than 80% and greater than 130% are unacceptable. The amounts of oxidized GSH are very similar in all experiments showing that GSH is not oxidized during the extraction procedures. Oxidation of GSH during extraction must be minimal.

4. Subject the extract to centrifugation (15 min, 4°C, 18,000^), and the supernatant fractions are used for the following assay procedures.

5. Determine the protein content of each supernatant fraction using a standard Bradford assay.

GLDH is bound to the inner mitochondrial membrane and Triton X-100 can be added if required to liberate the enzyme into the soluble supernatant fraction. Its inclusion is not necessary when ascorbate oxidase is assayed, even though this enzyme is bound to the cell wall. Triton X-100 also interferes with the following assays and should be avoided. High salt can be used to liberate cell wall-bond ascorbate oxidase (37).

GLDH (EC 1.3.2.3) activity is assayed by the coupled reduction of cytochrome c, which leads to a specific change in ODs 50 as described by Bartoli et al. (52).

1. Make the reaction buffer up fresh daily with 50 mM TRIS-HCl (pH 8.0), 60 pM cytochrome c, 3 mM galactono lactone (see Note 4), and 1 mM KCN to inhibit the reoxidation of the reduced cytochrome c (Fig.14.2).

2. Initiate the reaction by the addition of supernatant sample.

Spike, 40 nmol

Sample

Spiked before acidification

Spiked after acidification

3.3.2. Galactono-1, 4-Lactone Dehydrogenase Activity

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