A molecular basis for the inhibition of brain protein phosphatase 2A (PP2A) activity by oxidative stress was examined in a high-speed supernatant (HSS) fraction from rat cerebral cortex. PP2A activity was subject to substantial disulfide reducing agent-reversible inhibition in the HSS fraction. Results of gel electrophoresis support the conclusions that inhibition of PP2A activity was associated with the both the disulfide cross-linking of the catalytic subunit (PP2A(C)) of the enzyme to other brain proteins and with the formation of an apparent novel intramolecular disulfide bond in PP2A(C). Additional findings that the vicinal dithiol cross-linking reagent phenylarsine oxide (PAO) produced a potent dithiothreitol-reversible inhibition of PP2A activity suggest that the cross-linking of PP2A(C) vicinal thiols to form an intramolecular disulfide bond may be sufficient to inhibit PP2A activity under oxidative stress. We propose that the dithiol-disulfide equilibrium of a vicinal thiol pair of PP2A(C) may confer redox sensitivity on cellular PP2A.

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Reversible oxidation on proteins of vicinal thiols to form intraprotein disulfides is believed to be an important means by which redox sensitivity is conferred on cellular signaling and metabolism. Affinity chromatography using immobilized phenylarsine oxide (PAO), which binds preferentially to vicinal thiols over monothiols, has been used in very limited studies to isolate the fraction of cellular proteins that exhibit reversible oxidation of vicinal thiols to presumed disulfide bonds. A challenge to the use of PAO-affinity chromatography for isolation of readily oxidizable vicinal thiol proteins (VTPs) has been the lack of a disulfide reducing agent that reverses oxidation of the PAO-binding protein thiols and maintains these in the reduced state necessary to bind PAO but does not also compete with the VTPs for binding to the immobilized PAO. The present study demonstrates that the capture from a detergent-soluble rat brain extract of VTPs by PAO-affinity chromatography was improved greatly by use of the reducing agent tris(2-carboxyethyl)-phosphine which, unlike more traditional disulfide-reducing agents, does not contain a thiol group. Moreover, we show that, while a substantial fraction of total brain proteins contain PAO-binding thiols, only a fraction of these were readily and reversibly oxidized. The two most abundant of these redox-active proteins were identified as albumin and triose phosphate isomerase (TPI). We propose that TPI is a candidate intracellular redox receptor protein. The improved PAO-affinity method detailed here should enable the discovery of lower abundance novel redox-active regulatory proteins.

Our earlier finding that the activity of protein phosphatase 2A from rat brain is inhibited by micromolar concentrations of the dithiol cross-linking reagent phenylarsine oxide (PAO) has encouraged the hypothesis that the catalytic subunit (PP2Ac) of PP2A contains one or more pairs of closely-spaced (vicinal) thiol pairs that may contribute to regulation of the enzyme. The results of the present study demonstrate using immobilized PAO-affinity chromatography that PP2Ac from rat brain formed stable DTT-sensitive adducts with PAO with or without associated regulatory subunits. In addition, a subset of the PAO-binding vicinal thiols of PP2Ac was readily oxidized to disulfide bonds in vitro. Importantly, a small fraction of PP2Ac was still found to contain disulfide bonds after applying stringent conditions designed to prevent protein disulfide bond formation during homogenization and fractionation of the brains. These findings establish the presence of potentially regulatory and redox-active PAO-binding vicinal thiols on the catalytic subunit of PP2A and suggest that a population of PP2Ac may contain disulfide bonds in vivo.

uptake of neurotransmitters from the synapse and the The effects of 1 uM concentrations of arachidonic extrusion of intraneuronal Ca 2/ (5). Na containing 10 mM Tris (pH 7.0) and 1 mM EDTA. A crude synaptosomal pellet was obtained as described previously (13). The crude synaptosomal pellet was washed three times by repeated resuspension in the homogenization buffer and recentrifugation for 20 minutes at 12,000 1 g. The washed pellet was subjected to ultracentrifuLipoxygenases are dioxygenases that catalyze a ster-gation (SW41Ti rotor) on a discontinuous Ficoll (Pharmacia LKB eo-(S-configuration) and position selective oxidation Biotechnology Inc., Piscataway, NJ) density gradient for 30 minutes of arachidonic (eicosatetraenoic) acid to hydroperoxy-at 100,000 1 g as described by Booth and Clark (14). The synaptosome fraction was collected at the 7.5 % and 12 % Ficoll interface, eicosatetraenoic acids (HPETES) (see reference 1 for diluted with homogenization buffer and isolated by centrifugation reveiw). Lipoxygenases that produce hydroperoxides for 20 minutes at 20,000 1 g. A synaptosomal membrane fraction predominantly at the 5, 8, 12 and 15 positions of arachi-was obtained by osmotic lysis of the synaptosomes. Specifically, the donic acid have been identified (2). In addition to the synaptosomes were resuspendend in 20 volumes of 5 mM Tris (pH oxidation reactions, lipoxygenases may also catalyze 8.1) containing 1 mM EDTA and incubated on ice for one hour as described earlier (15). Following three strokes in a hand-held glass/ the further metabolism of HPETES to hydroxy Teflon homogenizer, the synaptosomal membrane fraction was iso-(HETES) and epoxy (leukotriene A 4 ) acids (1). Despite lated by centrifugation of the lysate for 20 minutes at 20,000 1 g. The an understanding of the lipoxygenase reaction mecha-synaptosomal membranes were resuspended in a minimal volume of nisms (1), knowledge of the biological function of lipoxy-the lysis buffer and 30 ul aliquots (containing about 300-400 ug protein) were frozen a minimum of 2 hours at 020Њ C prior to resusgenase products is limited (see reference 2 for review).

Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.