The regulation of exogenous NAD(P)H oxidation in spinach (Spinacia oleracea) leaf mitochondria by pH and cations

摘要: Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low 7.0, even in the presence 1 mM-CaCl2. There a proportional increase apparent Km for with decreasing H+ concentrations, suggesting that protonated on 2′-phosphate group true substrate. Exogenous NADH oxidized by 6.9. Under low-cation conditions, EGTA or EDTA (both no effect Vmax. oxidation, although removal bivalent cations membrane surface chelators could be observed use 9-aminoacridine fluorescence. In contrast, under high-cation lowered about 50%, probably due better approach negatively charged negative than conditions. medium, increased 50% addition cations. This caused lowering size potential through charge screening. contrast other cations, La3+ inhibited possibly binding lipids essential oxidation. The varied 6-fold response changes potential, active site is hampered potential. results demonstrate leaf cell can regulate mitochondrial NAD(P)H several mechanisms: pH; cation concentration general; Ca2+ particular. also emphasize importance electrostatic considerations when investigating kinetic behaviour membrane-bound enzymes.