Table Ii

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

Component (final concentration)"

1. 0.1-5.0 mM diamide (250 pM is recommended for routine use)

4. 800 ng of human recombinant PKCa

" Components presented in order of addition.

Method of addition

Added as 5 ¿¿1 of a diamide stock solution freshly made from the solid in 20 mM Tris-HCl, pH 7.5 Added as 5 ¡xl of a 2.1 mM GSH stock solution in

20 mM Tris-HCl, pH 7.5 To 105 (final volume) Present in equilibration buffer (95 fx 1)

the PKCa S-glutathiolation reaction, placed on ice, and then assayed for PKCa activity, as described below.

To analyze the DTT reversibility of PKCa inactivation, 30 mM DTT (final concentration) is added to the preincubation mixtures at the end of the 5-min preincubation period at 30°; the preincubation mixture volumes are adjusted to 110 Ml by the addition of 5 /¿I of either 20 mM Tris-HCl, pH 7.5 (—DTT samples), or 660 mM DTT in Tris buffer (+DTT samples) at 30°. The isozyme is then further preincubated at 30° for 5-20 min, placed on ice, and assayed for PKCa activity. An incubation period of at least 20 min in the presence of 30 mM DTT is generally necessary to fully reverse S-glutathiolation-mediated PKCa inactivation.4

Numerous assay procedures, including commercial assay kits, are available to measure PKC activity and can be used for the analysis of PKCa inactivation by S-glutathiolation. We have employed an assay that measures the transfer of 32P from [y-32P]ATP to a synthetic peptide substrate. The assay mixture components are shown in Table III. Addition of [y-32P]ATP to the assay mixtures initiates a 5- to 15-min phosphotransferase reaction at 30° with linear kinetics, and the reaction is terminated by the widely used practice of pipetting an aliquot of the assay mixture (40 /zl) onto an approximately 1-in2 piece of P-81 phosphocellulose paper (Fisher Scientific, Pittsburgh, PA), which binds the [32P]phosphopeptide product but not [y-32P]ATP. After the reacted mixture is pipetted onto each filter paper, the paper (numbered in pencil) is dropped into a 1-liter beaker filled with deionized water. The papers are collectively washed by decanting and refilling the beaker three additional times, and then counted in 5 ml of scintillation fluid.

C. Demonstrating S-Glutathiolation of Purified PKCa

1. Principle. The demonstration that diamide induces GSH-dependent and DTT-reversible PKCa inactivation (see Section II,B) points to PKCa S-glutathiolation as the inactivation mechanism for the following reasons. The involvement

TABLE III Composition of PKCa Assay Mixtures

Component (final concentration)"

Method of addition

Phosphatidylserine (PS), 30 /ig/ml

20 mM Tris-HCl (pH 7.5, 20 m Ai), 10 m M MgCl2, 0.2 m M CaCl2 (or 1 mM EGTA) 10 /àM [Ser-25]PKC( 19-31 ) peptide substrate Water

PKCa, 80 ng 6 /¿Ai [)/-32P]ATP (50008000 cpm/pmol)

10 [iI of a sonicated dispersion of 0.36-mg/ml bovine brain PS (>98% pure) in water; not present in assays of basal activity

20 ¡A of a 6x stock solution; EGTA is present in assay tubes that measure basal activity

10 /¿I of a 120 ¡iM peptide stock solution (peptide is purchased from Peninsula or BACHEM Bioscience)

To adjust final volume to 120 /¿I

10 /il of preincubated PKCa

10 fil of 72 nM [y-32P]ATP is added to initiate the reaction. Reactions are initiated in tubes at 30° at 15-sec intervals and terminated 5-15 min later at 15-sec intervals

" Components presented in order of addition.

of the oxidant activity of diamide in the inactivation mechanism and the reversal of inactivation by DTT both indicate a role for disulfide bridge formation, and a requirement for GSH is also evident. The only simple inactivation mechanism that can account for these mechanistic features is PKCa S-glutathiolation [Eq. (1)].

Diamide

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