Uvmc2 spectrophotometer

The activities of the mitochondrial OXPHOS complexes were measured at 37°C with a UVmc2 spectrophotometer (SAFAS, Monaco). Activity of the NADH ubiquinone reductase (CI), succinate ubiquinone reductase (CII), ubiquinol cytochrome C reductase (CIII), cytochrome C oxidase (CIV) and citrate synthase (CS) were measured according to standard methods [28 (link), 29 (link)]. NADH ubiquinone reductase (NUR) activity was assayed in KH2PO4 buffer (50mM, pH 7.5), containing 3.75 mg/ml fatty acid-free BSA and 0.1mM decylubiquinone. 0.1 mM NADH was added to initiate the reaction. Parallel measurements in presence of Rotenone (5μM) were used to determine the background rate. NADH:FeCN and NADH:HAR reductase activities were assayed in 20 mM KH2PO4 buffer (pH 7.5), with substrate and acceptor concentrations as previously described [30 (link)]. Background rates (without CI) were controlled for each experiment and RSV concentration. NADH:FeCN, NADH:HAR and NADH:Decylubiquinone oxidoreductions were monitored at 340 nm (ε = 6.22mM–1.cm–1).

Activities of complexes I, II, and SDH (succinate dehydrogenase) of the respiratory chain were determined at 37 °C with an UVmc² spectrophotometer (SAFAS, Monaco) on mitochondrial fractions [25 (link)].

The specified activities of OXPHOS complexes were measured at 37°C on a UVmc2 spectrophotometer (SAFAS, Monaco) in a mitochondrial-enriched fraction from frozen cell pellets. The activities of NADH ubiquinone reductase (complex I, CI), ubiquinol cytochrome c reductase (complex III, CIII), F0F1-ATP hydrolase (complex V, CV) and citrate synthase (CS) were measured according to routine methods.²² (link)^,25 (link)

Classical oenological parameters methods were measured following International Organisation of Vine and Wine (OIV) reference methods. Alcoholic percentage was determined by Fourier transformed infrared spectroscopy—FTIR (WineScan, FOSS France, Nanterre, France) (OIV-MA-BS-08) [31 ], free and total sulfur dioxide by the automated iodometric method (Titromatic, Crison Instruments, Alella, Spain (OIV-MA-AS323-04B) [32 ], and pH by the potentiometric method (pH meter Consort C3010, Consort bvba, Belgium) (OIV-MA-BS-13) [33 ]. Finally, the Total Polyphenol Index (TPI) was determined as absorbance at 280 nm using a UV mc2 spectrophotometer (Safas, Monaco) [29 ].

The activities of the mitochondrial OXPHOS complexes were measured at 37 °C on a UVmc2 spectrophotometer (SAFAS, Monaco, Monaco) in a mitochondrial-enriched fraction from frozen cell pellets. The activities of NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol cytochrome C reductase (complex III), cytochrome C oxidase (complex IV) and citrate synthase (CS) were measured according to the method described by Desquiret-Dumas et al. [22 (link)].

Hydratase activity was monitored at 263 nm using a UVmc2 spectrophotometer (SAFAS) in the presence of trans-2-dodecenoyl-CoA (C_12:1-CoA; ∆A of 0.67 for a variation of concentration of 100 µM) synthesized as previously described^{48 (link)}. Kinetic assays were performed in a quartz cuvette for 40 sec at room temperature, in 100 mM sodium phosphate buffer pH 7.0 in the presence of 10 µM C_12:1-CoA. After equilibration of the baseline, reactions were started by adding purified 50–200 nM H-HadD protein. Control experiments lacking the protein were realized.