Briefly, freshly isolated mitochondria were added to reaction buffer (0.6 M mannitol, 0.1 mM EGTA, 2 mM MgCl2, 10 mM KPi, 5 mM α-ketoglutarate, 0.01 mM Ap5A, 10 mM Tris-HCl, pH 7.4) containing the ATP detection system (2.5 mM glucose, hexokinase (2 E.U ., glucose-6-phosphate dehydrogenase (2 E.U ., 0.2 mM NADP).
For these measurements, purified OpuA in the presence of 0.05% (w/v) n-dodecyl-β-maltopyranoside (DDM) was mixed such that the protein (at a final concentration of 100 µg/mL) was in a buffer composed of 10, 50 or 250 mM KPi pH7.0, 20% glycerol and 0.05% DDM.
The sample was then dialyzed in 300 mM KCl, 20 mM KPi, 2 mM MgCl2 (pH 7) column buffer.
The purified enzymes were incubated at 50 64 °C at the same enzyme concentration (9.1 × 10−5 mg/ml) with 25 mM KPi pH 8.0, 5 mM MgCl2, 0.1 mg/ml BSA and 100 mM glucuronic acid.
Cell pellets were resuspended in 300 μL of 50 mM KPi, 100 mM glucose, 10 mM substrate, pH 7.0.
Briefly, frozen skeletal muscle tissue samples (25 35 mg) were hand homogenized in 25 µl of phosphate buffer (50 mM Kpi, 5 mM EDTA, 0.5 mM DTT, 1.15%KCl (w/v)) per milligram of tissue.
To the sample of 5 μM CcO and 110 mM spin trap POBN [α- 4-pyridyl-1-oxide)- N- tert-butylnitrone] was added 200 μM H2O2 [α- 4-pyridyl-1-oxideand 0.1% DM at 23 °C].
Cells, grown in the respective media, were harvested at OD600≈0.5 and washed with 0.5 mM KPi, pH 6.4, 70 mM KCl plus 1 mM MgSO4.
According to the above and using (3) and (4), one can easily find {m}_L=frac{Q^{-1/2}}{2{gamma}^2}{displaystyle underset{0}{overset{+infty }{int }} dkfrac{c{ h}^{-1} kpi /2}{f_k}}.
In this case, calculating variations, taking into account (2), Lagrangian (1) and solving the corresponding variational problem for v L < ω M Λ, we find for the DW gyrotropic bending: qleft xi right)=frac{v}{omega_MvarLambda}varDelta {displaystyle underset{0}{overset{+infty }{int }} dkfrac{ cos left( kxi /varLambda right) c{h}^{-1} kpi /2}{f_k}}, (3).
Table 4 Field tests results Test Duration Missed alarms False alarms Tuning phase 50 tests 0.05 0.12 KPI tests 20 tests 0. 0.05 Live Demo 30 tests 0.02 0.02.
