The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg2+-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na2SO3-dependent Mg2+-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg2+-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg2+-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg2+-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen.The active state decayed in the dark with a t%,. of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, oiodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.Activity of the proton-ATPase of spinach and pea chloroplasts is light regulated. The membrane-bound enzyme is activated after a period of illumination and it decays back to a latent state in the postillumination darkness. Light modulation (activation/deactivation) has been demonstrated with thylakoids isolated from illuminated leaves, protoplasts, and freshly lysed intact chloroplasts (12,13,15,19,22,26
MATERIALS AND METHODSMaize plants (Pioneer hybrid No. 3747) were grown in a greenhouse with a 15-h photoperiod maintained with supplemental low pressure sodium lamps and incandescent lamps. Intact chloroplasts and thylakoids were isolated from the tips of fully expanded second and third leaves of 7-to 14-d-old plants. The intact mesophyll chloroplasts were isolated by the procedure of Jenkins and Russ (7) as modified by Thompson et al. (25). The chloroplasts were judged to be 85 to 95% intact based on the FeCy penetration test (10). Maize mesophyll thylakoids were isolated in the following way: 50 g of leaf tips were chopped into 0.5-1.0 cm segments. The segments were homogenized with a Polytron PT-35 at a setting of '6' for 5 s in 300 mL of a medium containing: 400 mm sorbitol, 50 mm Tricine-Na+ (pH 7.8), 10 mM NaCl, 2 mM MgCl2, 0.2% BSA, 0.2% ascorbate, plus 2 g of polyclar AT. The homogenate was filtered through a combination of cheesecloth and miracloth and centrifuged for 5 min at 3000g to collect the thylakoids. The thylakoids were washed once (3000g)...