A Proposed Mechanism for the Stimulatory Effect of Bicarbonate Ions on ATP Synthesis in Isolated Chloroplasts

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The rate of nouphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in weD-coupled (ATP/2e-= 0.9-1.1) maize mesophyD choroplasts Is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin andHeg ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations In medium pH, and to the presence of adenine nucleotides and energy transfer inhdbitors in the assay medium Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3-to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in Cs and C4 mesophyll chloroplasts.A number of recent studies have examined the relationship between proton flux through the coupling factor complex (CFo-CF1) and electron flow in chloroplasts (2, 7-11, 24, 25, 27-29). In C3 mesophyll chloroplasts which have developed a large transmembrane proton concentration gradient, there appears to be a pathway for proton efflux through CFo-CF, that is not coupled to ATP synthesis (8,11,17,19,24,27,28 (8,17,24,27,28). Studies of the effects of energy transfer inhibitors, e.g. phlorizin, alkyl tins, Hg2+ ions, etc.on basal electron flow also suggest a role for CFo-CF, in the control of proton leakage from the intrathylakoid space via this pathway (8,11,27,28). Our aim was to compare the effects of adenine nucleotides and energy transfer inhibitors on the rate of nonphosphorylating electron flow and on proton fluxes in mesophyll chloroplasts isolated from both a C3 plant (pea) and a C4 plant (maize).

Section: Discussionmentioning

confidence: 99%

The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts

Cole

Macpeek²,

Cohen³

1981

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“…The assays were performed at 37°C for 10 min. ATP synthesis was assayed as described elsewhere (3,4). Pi was estimated colorimetrically (21).…”

Section: Methodsmentioning

confidence: 99%

Sulfhydryl Reagents and Energy-Linked Reactions in Monocot Thylakoids

Cohen

Baxter²

1990

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Monofunctional maleimides have been used to covalently modify the coupling factor protein of monocot thylakoid membranes. As with dicot thylakoids, incubation of the monocot thylakoids with maleimides in the light but not in the dark results in inhibition of both ATP synthesis and hydrolysis. In the dark, sites on the Y and subunits of maize Zea mays coupling factor 1 are modified after incubation of maize mesophyll thylakoids with the fluorescent maleimide N-(anilinonaphthyl-4) maleimide. A light accessible site localized solely to the y subunit has also been demonstrated. itaria sanguinalis]) with bifunctional maleimides or thiol oxidants in the light does not result in functional uncoupling, i.e the bifunctional reagents act more like energy transfer inhibitors. The lack of functional uncoupling could be due either to a failure of the reagents to cross-link key sulfhydryl residues in the y subunit or to the continued ability of the y subunit to gate proton movements through the chloroplast coupling factor complex even though its conformation has been altered by sulfhydryl reagents.The y subunit of the spinach proton-ATPase contains one disulfide bridge and two free sulfhydryls (9). One of the free sulffiydryls (S4 in McCarty's terminology [15]) is readily accessible to modification by maleimides in the dark. Modification of S4 has no apparent effect on energy transduction mediated by the coupling factor (CF,3). The second free sulfhydryl (S3) is not readily accessible, but becomes available when the thylakoid membrane is energized. Modification of S3 with maleimides inhibits both ATP synthesis and hydrolysis without affecting proton leakiness (10, 12 (6,20). When S3 (Cys-89) and S4 (Cys-322) of spinach y are

“…as described by Anthon and Jagendorf (1). ATP synthesis was assayed as described elsewhere (6). Pi was estimated colorimetrically (24).…”

Section: Methodsmentioning

confidence: 99%

Characteristics of the Mg²⁺-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

Cohen

1989

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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)...