The effect of calcium and calmodulin inhibitors on NADH oxidation by isolated plasma membrane vesicles preloaded with NADH

Abstract Right-side-out plasma membrane vesicles of soybean ( Glycine max (L.) Merr.) hypocotyl were loaded with NADH by electroporation. After washing it was possible to measure a decrease of NADH within the vesicles. Addition of the membrane impermeant electron acceptor hexacyanoferrate III to the experimental solution caused an increase of NADH oxidation, while the acceptor was reduced. NADH oxidation and reduction of the external acceptor were influenced by calcium and by calmoudin inhibitors. Ca 2+ up to 100 μM stimulates the NADH:hexacyanoferrate III oxidoreductase activity, while millimolar concentrations inhibit. The stimulation is Ca 2+ specific and sensitive to calmodulin inhibitors. The membrane impermeant inhibitor, calmidazolium, is by far the strongest one, while the permeant compounds fluphenazine, trifluoperazine and W7 are less active. Compound W5 turned out to be practically inactive.