Oxidoreductase activity of the CLIC proteins.

<p>Oxidoreductase enzymatic activity was measured using 5 uM of CLIC proteins or HcTrx-5 or Grx-1, 250 uM NADPH, 1 mM HEDS and 50 nM GR. The reaction was initiated by the addition of 1 mM GSH and the absorbance of NADPH was monitored at A_{340 nm}. Reaction conditions: 5 mM potassium phosphate with 1 mM EDTA, pH 7, at 37°C. (<b>A</b>) Activity of CLIC1, CLIC2 and CLIC4 compared to HcTrx-5 and Grx-1 (positive controls). (<b>B</b>) Activity of 5 uM CLIC1 dimer compared to 5 uM CLIC1 monomer. <i>Error bars</i> represent the S.E. of at least three independent measurements.</p

Glutathione-dependant activity of the CLIC proteins.

<p>(<b>A</b>) The reaction mixture contained 2 mM EDTA in 0.1 M Tris-HCl (pH 7.5), 5 uM reduced CLIC1, CLIC2 or CLIC4 (WT) protein, 200 uM NADPH, 750 uM HEDS, 50 nM TrxR and 5 uM Trx-1 (included as a positive control). (<b>B</b>) Insulin disulfide reductase assay to determine catalytic activity of Trx-1 and CLIC1 based on solution turbidity monitored by A_{650 nm} over 30 minutes.</p

Conserved G-site motif in members of the CLIC family.

<p>Multiple sequence alignment of human proteins: CLIC 1-6, GST-omega and Grx1-3. Highlighted in grey is the glutaredoxin/thioredoxin active site motif (G-site) (Accession numbers: CLIC1 (CAG46868), CLIC2 (CAG03948), CLIC3 (CAG46863.1), CLIC4 (CAG38532), CLIC5 (AAF66928), CLIC6 (NP_444507), GST-omega (AAF73376), Grx-1 (BAAO4769), Grx-2 (AAK83089) and Grx-3 (AAH0528289) obtained from ClustalW.</p

Effect of chloride ion channel inhibitor drugs on the oxidoreductase enzymatic activity of CLIC1.

<p>5 uM of CLIC1 reduced (WT) or HcTrx-5 protein was incubated with 10 uM IAA-94, A9C, DIDS or Saxitoxin for ∼1 hour prior use of the protein in the assay. The enzyme assay mixture contained 250 uM NADPH, 1 mM HEDS, 50 nM GR in 5 mM potassium phosphate buffer with 1 mM EDTA, pH 7, at 37°C. The consumption of NADPH was monitored at A_{340 nm} post addition of 1 mM GSH. <i>Error bars</i> shown represent the S.E. of at least three experimental measurements.</p

Comparison of the oxidoreductase activity of CLIC1 (WT) monomer and CLIC1-Cys mutants.

<p>(<b>A</b>) The reaction contained 1 mM EDTA in 5 mM potassium phosphate (pH 7), 250 uM NADPH, 50 nM GR, 1 mM HEDS and 5 uM of reduced CLIC1 (WT), CLIC1-C24A, CLIC1-C24S or CLIC1-C59A. The mixture was incubated for 5 mins at 37°C before initiation of the reaction with the addition of 1 mM GSH followed by monitoring NADPH absorbance at A_{340 nm}. <i>Error bars</i> represent the S.E. of at least three experimental repeats. (<b>B</b>) A reaction of 5 uM of CLIC1 (WT) reduced monomer or CLIC1-C59A protein, 250 uM NADPH, HEDS (0, 0.25, 0.5, 1, 2, 4 or 6 mM) and 50 nM GR. The reaction was initiated by the addition of 1 mM GSH and the absorbance of NADPH was monitored at A_{340 nm}. The reaction conditions where 5 mM potassium phosphate with 1 mM EDTA, pH 7, at 37°C.</p

Effect of cholesterol on the enzymatic activity of CLIC1.

<p>5 uM of CLIC1 monomer (WT) protein was incubated with 0.4, 0.8 and 1.6 mM cholesterol for ∼1 hour prior to its addition to a reaction mixture of 250 uM NADPH, 1 mM HEDS, 50 nM GR in 5 mM potassium phosphate buffer with 1 mM EDTA, pH 7, at 37°C. The consumption of NADPH was monitored at A_{340 nm} post addition of 1 mM GSH. Control included all the reaction components including 1.6 mM cholesterol, except with no CLIC1 protein. The <i>error bars</i> shown represent the S.E. of at least three experimental measurements.</p

Sodium selenite and dehydroascorbic acid as substrates for CLIC1.

<p>(<b>A</b>) The oxidoreductase enzymatic reaction using sodium selenite as a substrate was performed in 0.1 mM Tris-HCl (pH 7.5) with 1 mM EDTA containing 200 uM NADPH, 50 nM GR,15 uM sodium selenite, 0.1 mg/mL BSA and 5 uM CLIC1(WT) reduced monomer or 5 uM Grx-1 as a control. The reaction was initiated by the addition of 50 uM GSH at 20°C with consumption of NADPH measured at A_{340 nm}. <i>Error bars</i> represent the S.E. of at least three experimental repeats. (<b>B</b>) The reaction was performed in 0.1 mM Tris-HCl (pH 7.5) with 1 mM EDTA containing 200 uM NADPH, 50 nM GR, 5 uM CLIC1 (WT) reduced monomer and sodium selenite (0, 1, 2, 4, 8 or 16 uM). The initiation of the reaction was achieved by adding 50 uM GSH at 20°C where the consumption of NADPH was measured at A_{340 nm}. (<b>C</b>) The oxidoreductase enzymatic reaction using DHAR as a substrate was performed in 137 mM sodium phosphate buffer (pH 7.5) containing 2 mM EDTA, 0.35 mM NADPH, 50 nM GR, 2 mM GSH and 1 mM DHA. The reaction was initiated after addition of 5 uM reduced CLIC1, CLIC4 or HcTrx-5 (as control). Consumption of NADPH was measured at A_{340 nm}. <i>Error bars</i> represent the S.E. of at least three experimental repeats. (<b>D</b>) DHAR activity of the CLIC proteins was determined using 137 mM sodium phosphate buffer (pH 7.5) with 2 mM EDTA, 0.35 mM NADPH, 50 nM GR, 2 mM GSH and DHA (0, 0.25, 0.5,1, 2, 4 or 6 uM). The reaction was initiated after the addition of 5 uM CLIC1 (WT) protein and the NADPH consumption was monitored at A_{340 nm}.</p