Oxidation rates were determined polarographically with a Clark electrode (Rank Brothers, Cambridge, UK). The use of a combined enzymatic system composed of glucose (5 mM), hexokinase (2.5 U mL−1 Sigma H4502), glucose-6-phosphatase dehydrogenase (2.5 U mL−1, Sigma G6378), NADP+ (1.6 mM) allowed the determination of phosphorylation rate since the NADPH production by this system is stoichiometrically linked to mitochondrial ATP synthesis rate. Changes in the rate of NADPH production were monitored at 340 nm by placing an optic fiber connected to a spectrophotometer in the oxygraphic vessel (Cary 50; Varian, Grenoble, France). Conversion of NADPH absorbance into NADPH concentration was performed using a molar extinction coefficient of 6.22 mM−1 cm−1. Bioluminescent assays were used to measure both ADP and ATP concentration during each experiment (Gouspillou et al., 2011 (link)). This method allowed the determination of the oxidation (KmVox) and phosphorylation (KmVp) rates affinities for ADP, as well as the determination of mitochondrial coupling efficiency as the ratio of phosphorylation over oxidation rates.
Glucose 6 phosphatase dehydrogenase
Manufactured by Merck Group
Sourced in China
Glucose-6-phosphatase dehydrogenase is an enzyme that catalyzes the first step in the pentose phosphate pathway. It oxidizes glucose-6-phosphate to 6-phosphoglucono-δ-lactone, generating NADPH in the process.
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3 protocols using glucose 6 phosphatase dehydrogenase
1
Mitochondrial Oxidation and Phosphorylation Rates
2
Measuring HO-1 Activity in Cell Lysates
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HO-1 activity was measured as previously described with minor modification [23] (link). Cell lysates were added to the reaction mixture containing 30 μM haemin (Sigma–Aldrich), 2 mg/ml of rat liver cytosol, 1 mM Mgcl2, 3 units of glucose-6-phosphatase dehydrogenase (Sigma–Aldrich), 1 mM glucose-6-phosphate (Solarbio Science & Technology, Beijing, China), 2 mM reduced nicotinamide adenine dinucleotide phosphate (Beyotime Institute of Biotechnology, Jiangsu, China) and 0.1 M potassium phosphate buffer. The reaction was conducted at 37°C in the dark for 30 min and terminated by the addition of 1 ml of chloroform. The concentration of bilirubin was determined as the difference between the concentrations measured at 464 nm and 530 nm. The results were expressed as picomoles of bilirubin per milligram of protein.
3
Bilirubin Production Assay in Cells
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HO activity was measured as previously described (Vanella et al. 2010) with minor modification (Fig. 1A,C). Cell homogenates were added to a reaction mixture containing 30 μM hemin (Sigma-Aldrich), 2 mg/ml rat liver cytosol, 1 mM Mgcl 2 , 3 unit glucose-6-phosphatase dehydrogenase (Sigma-Aldrich), 1 mM glucose-6-phosphate (Roche, Basel, Switzerland), 2 mM NADPH (Beyotime), and 0.1 M potassium phosphate buffer (pH 7.4). The reaction was conducted at 37°C in the dark for 30 min and terminated by addition of 1 ml of chloroform. The extracted bilirubin was defined as the difference between 464 and 530 nm (ε = 40 mM -1 cm -1 ). The results were expressed as nmol of bilirubin/5 × 10 6 cells/h.
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