Table Iv

Preincubation Conditions Required to Demonstrate PKCa 35S-Glutathiolation by Nonreducing SDS-PAGE

Condition

Purpose

To establish that PKCa is not covalently labeled by [35S]GSH in the absence of an oxidant. Corresponds to noninactivating conditions

To demonstrate oxidant-induced covalent labeling of PKCa by

[35S]GSH. Corresponds to inactivating conditions To establish a close correlation between PKCa inactivation and 35S-glutathiolation, a range of diamide concentrations should be employed To demonstrate that the covalent [35S]GSH labeling of PKCa in step 2 is due to PKCa S-glutathiolation, based on the DTT reversibility of the labeling. PKCa is preincubated with [35S]GSH + diamide followed by further incubation with DTT. Corresponds to noninactivating conditions experiments that the shift is indeed due to protein S-glutathiolation. To measure the stoichiometry of the covalent incorporation of [35S]GSH into PKCa, [35S]GSH-modified PKCa can be precipitated with trichloroacetic acid (TCA) and captured on filter paper4 as described below.

2. Method. To detect PKCa 35S-glutathiolation by nonreducing SDS-PAGE and autoradiography, the preincubation mixtures employed in the analysis of PKCa inactivation (Table II) are modified as follows. A 100 [35S]GSH (-125 mCi/ mmol; DuPont-NEN, Boston, MA) concentration is substituted for 100 ¡iM GSH, and the DTT concentration is reduced to 10 mM, because higher DTT concentrations may produce dark backgrounds in the autoradiograms. PKCa preincubation mixtures that correspond to the three sets of conditions outlined in Table IV are required to demonstrate PKCa 35S-glutathiolation. At the end of the 5-min preincubation period at 30°, an equal volume of 2x nonreducing SDS-PAGE sample buffer [0.125 MTris-HCl (pH 6.8), 4.0% (w/v) SDS, 20% (v/v) glycerol, 0.002% (w/v) bromphenol blue] is added to each preincubation mixture, and the samples are boiled for 3 min. The samples are analyzed by nonreducing 10% (w/v) SDS-PAGE (80-120 ng of PKCa per lane). [Gels with 7.5% (w/v) polyacrylamide are recommended for the alternative approach of monitoring the migration position shift of PKCa by Western analysis.] The gel is stained with Coomassie dye to detect molecular weight markers for subsequent determination of the migration positions of bands detected on the autoradiogram. Next, the gel is washed with deionized water and impregnated with Amplify fluorographic reagent (Amersham Pharmacia Biotech, Piscataway, NJ) by shaking the gel for 15 min at room temperature in the presence of sufficient Amplify solution to cover the gel. By converting

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