Table I

Preincubation Conditions for Analysis of PKCa Inactivation by Diamide-Induced S-Glutathiolation

Condition Purpose

1. PKCa alone Positive control that represents 100% PKCa activity

2. With diamide Control to measure weak or no PKCa inactivation by diamide alone

3. With GSH Control to demonstrate lack of effect of 100 ¡iM GSH

on PKCa activity

4. With GSH + diamide Conditions that support potent PKCa inactivation by S-glutathiolation

The potent induction of GSH-dependent, DTT-reversible inactivation of PKCa by diamide, coupled with the direct demonstration of the S-glutathiolating modification of the isozyme in association with the inactivation (which is described in Section II,C), is indicative of PKCa inactivation by S-glutathiolation. This is because the only other inactivation mechanisms possible, intramolecular disulfide bridge formation within PKCa and intermolecular disulfide bridge formation between PKCa molecules, are excluded by the inability of diamide alone to potently inactivate the isozyme.4 The experimental conditions and procedures described below produce potent PKCa inactivation by S-glutathiolation, which is measured as GSH-dependent, diamide-induced isozyme inactivation.4 In an analysis of recombinant human PKCa, these conditions resulted in a loss of >80% of the activity of the isozyme by a GSH-dependent and DTT-reversible, diamide-induced mechanism across abroad range of diamide concentrations (0.1-5.0 mM). Diamide alone inactivated the isozyme weakly or not at all, depending on the diamide concentration, and GSH alone (100 (jlM GSH) had no effect on PKCa activity.4 It is worthwhile to note that millimolar GSH concentrations should be avoided, as non-redox-inhibitory effects against purified PKC isozymes have been observed at GSH concentrations in the range of 1-10 mM.n The wide range of diamide concentrations achieving near-full PKCa inactivation by an S-glutathiolation mechanism indicates the flexibility of the system and thus its potential for detecting PKCa inactivation by S-glutathiolation when the conditions are modified to tailor the experiments to specific applications.

2. Method. To demonstrate diamide-induced PKCa inactivation by S-glutathiolation, a PKCa sample with refreshed thiols (see Section II,A) is prein-cubated under the four conditions specified in Table I. Preincubation mixtures containing the components indicated in Table II (105 /¿I in capped 0.2-ml tubes) are vortexed (briefly, to minimize aeration), preincubated for 5 min at 30° to support

12 N. E. Ward, D. S. Pierce, S. E. Chung, K. R. Gravitt, and C. A. O'Brian, J. Biol. Chem. 273,12558 (1998).

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