valinomycin and lanthanum-chloride

Free Mg(2+) in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg(2+) concentration ([Mg(2+)]) in chloroplasts has been lacking. We measured internal chloroplast [Mg(2+)] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal [Mg(2+)] was estimated to be 0.50 mM, and illumination caused an increase in [Mg(2+)] to 2.0mM in the stroma. The light-induced increase in [Mg(2+)] was inhibited by a blocker of driven electron transport and uncouplers. The K(+)-specific ionophore valinomycin inhibited the [Mg(2+)] increase in the absence of external K(+), and addition of KCl restored the [Mg(2+)] increase. NH(4)Cl, which induces stromal alkalinization, enhanced the [Mg(2+)] increase. A Ca(2+)-channel blocker, ruthenium red, inhibited the [Mg(2+)] increase, but LaCl(3) had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in [Mg(2+)]. This system for measuring internal chloroplast [Mg(2+)] might provide a suitable system for assay of Mg(2+) transport activity of chloroplast membranes.

Topics: Calcium; Calibration; Chloroplasts; Dose-Response Relationship, Drug; Fluorescent Dyes; Fura-2; Ionophores; Lanthanum; Light; Magnesium; Potassium Chloride; Ruthenium Red; Spectrometry, Fluorescence; Spinacia oleracea; Time Factors; Valinomycin

Raman spectroscopy (RS) study shows that the "potential sensor" responds to changes in intramembrane potential by conformational changes. The mechanism of regulation of the channel "gate" by the carotenoid potential sensor is discussed.

Topics: Animals; beta Carotene; Calcium Chloride; Carotenoids; Egtazic Acid; In Vitro Techniques; Ion Channels; Lanthanum; Liposomes; Membrane Potentials; Potassium Chloride; Ranidae; Sciatic Nerve; Spectrum Analysis, Raman; Valinomycin