Heinrich Strotmann

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1).

1. [14C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF1. [14C]ADP binding exhibits a monophasic concentration curve with a Km of 2 micronM. 2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF1 conformation is established. 3. Re-binding of [14C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF1, [14C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF1 is converted to a non-exchangeable form. 4. Energy-dependent adenine nucleotide exchange on CF1 is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.

Inhibition of nitrate uptake by ammonia in a blue-green alga, Anabaena cylindrica

Ammonia at concentrations above 1×10-5 M inhibits uptake of nitrate in the nitrogen-fixing blue-green alga, Anabaena cylindrica. This inhibition takes place both in the light and in the dark. The rate of nitrate uptake is stimulated by light. Addition of relatively high concentrations of nitrate (1–10 mM) reversibly inhibits ammonia uptake. FCCP, an uncoupler of phosphorylation, inhibits both nitrate and ammonia uptake. Ammonia may inhibit nitrate uptake by reducing the supply of energy (ATP) for active nitrate transport.

Nucleotide binding and regulation of chloroplast ATP synthase

The CF,CF 1 complex catalyzes the reversible formation of ATP coupled to transmembrane proton translocation in chloroplasts. The enzymatic activity of membrane-bound and isolated CF, is however latent. In chloroplasts, induction of the ATP hydrolyzing activity requires pre-illumination of the membranes (light-triggered ATPase) [l-5]. Recently, it has been demonstrated that light activation is related to release of tightly-bound ADP from CF1 [6]. However, the induced ATPase is known to be deactivated by ADP [7]. This reaction is quantitatively related to re-binding of ADP to the previously depleted sites to form the tight CF l-ADP complex again [6]. Moreover, it has been shown that under different conditions the rate of ATP hydrolysis is controlled by the fraction of CF, molecules which are free from tightly bound ADP [6]. In ATP synthesis, energy-dependent activation of CF, is also most likely a prerequisite, as concluded from the induction and threshold phenomena in photophosphorylation [8]. Furthermore, pre-treatments of the chloroplasts which induce the ATP hydrolyzing activity of CF, increase the yield of ATP formation in single turnover flashes [9]. In [IO] it was suggested that the physiological activation process may be monitored by energy-dependent exchange of tightly bound adenine nucleotides on CF ,. Under phosphorylating conditions tightly bound adenine nucleotides are exchanged against medium ADP [I l-141. The energy-dependent reaction is the release step facilitated by a conformational change of

Determination of the H+ /ATP ratio of the H+ transport-coupled reversible chloroplast ATPase reaction by equilibrium studies

Fluorescence quenching of 9‐aminoacridine was followed to ascertain the state of light‐induced transthylakoidal Δ H+ at energetic equilibrium with a subsequently added mixture of ATP, ADP and phosphate. In the measured range, the logarithm of relative fluorescence quenching at equilibrium is a linear function of the imposed phosphate potential. The slope of the line is shown by mathematical deduction to be equal to the ATP/H+ stoichiometry of the H+‐coupled reversible ATPase reaction. Determination of the stoichiometry by this method neither relies on the standard phosphate potential nor on the internal thylakoid volume. The results confirm that during ATP synthesis or hydrolysis 3 H+ per ATP are translocated through the CF0‐CF1 complex.

Nucleotide specificity of CF1-ATPase in ATP synthesis and ATP hydrolysis

Coupling factor (CF,) from chloroplasts is known to catalyze the reversible formation of ATP from ADP and Pi. In chloroplasts and with the isolated enzyme no ATPase activity is observed unless the protein is activated. Activation can be achieved by artificial modification (trypsin treatment [ 11, heat [2], DTT [3]), which yields a Ca2+-dependent ATPase. Physiologically, the membrane-bound enzyme can be activated by light [4-71. Light-triggered ATPase is stimulated by thiol reagents. Most likely a lightdependent activation of CFr is also involved in the process of photophosphorylation [8-l 11. From the differential inhibition of partial reactions by Fab fragments of antibodies against CFr, it was concluded [ 121 that CFr contains two catalytic sites, one specialized for ATP synthesis and one for ATP hydrolysis and related reactions, like ATP-Pi exchange. The same was concluded [ 131 on the basis of studies with 1 ,N6-etheno analogs of ADP/ATP and CDP/CTP. The nucleoside diphosphates were found to replace ADP in phosphorylation, but the nucleoside triphosphates were only poor substitutes in the ATPase reactions. The occurrence of two separate sites responsible for the catalysis of the forward and back reaction, respectively, would be of unique interest in enzymology. Therefore, we re-investigated the problem by determining the nucleotide specificities of the reactions. To avoid misinterpretation, it is important to determine the kinetic parameters (Vmax, K,) under comparable conditions and to consider their meaning critically.

Effects of external factors on photophosphorylation and exchange of CF1-bound adenine nucleotides

Chloroplast coupling factor CFI contains firmly bound adenine nucleotides which were shown to be slowly exchanged in de-energized chloroplasts and in the isolated enzyme [1-9] . On energization of the thylakoids either by light-dependent electron transport [1-3,9] or by an acid-base transition [9], bound adenine nucleotides are rapidly replaced by free ADP or ATP. From aH incorporation studies [10,11 ], fluorescence studies of covalently bound fluorescamine [12], and other indirect evidence [13] it was concluded that energization induces a conformational change of CFI. Between the energy-dependent change of CF~ conformation and the alteration of adenylate binding an intrinsic relationship may be suggested. The functional role of bound adenine nucleotides in the mechanism of photophosphorylation has recently been interpreted [3,7] by analogy with the conformational hypothesis of oxidative phosphorylation [14,15]. In this concept the actual energyrequiring step of the phosphorylation cycle was thought to be the transition of CFt from an inactive to an adenylate-exchangeable form. By this reaction preformed bound ATP was assumed to be released and replaced by free ADP. The formation of tightly bound ATP itself was believed to be energy-independent. Assuming that the dissociation equilibria of CFI-ADP and CFI-Pi complexes were high in comparison to CF~-ATP complex in the inactive state, the equilibrium of the ATPase reaction was predicted to be shifted towards the formation of firmly bound ATP [3]. In the present paper light-induced exchange of bound adenine nucleotides was studied as a function of several external parameters. The results indicate a corresponding behavior of adenylate exchange and photophosphorylation in dependency of light intensity, pH, uncoupling, and electron transport inhibition. On the other hand, arsenate and phlorizin which are known to inhibit photophosphorylation, do not affect the light-dependent exchange of bound adenine nucleotides. Mg 2÷ ions stimulate both, photophosphorylation and adenylate exchange, however in a different way.

Control of ATP hydrolysis in chloroplasts

Dark ATP hydrolysis catalysed by the membrane‐bound preactivated thiol‐modified chloroplast ATPase was measured at constant initial ATP concentration and constant initial phosphate potential c ATP/c ADP·cP i which was adjusted by inverse variation of the concentrations of ADP and Pi. Under these conditions, the rate of ATP hydrolysis is strongly inhibited as the concentration of ADP is increased and the concentration of Pi is decreased. Inhibition is preferentially caused by ADP‐dependent inactivation of ATPase molecules. At low initial ADP concentration, the transmembrane proton gradient generated by effective ATP hydrolysis protects the enzyme from deactivation in spite of progressive accumulation of ADP because energy‐dependent release of ADP counteracts its binding. Deactivation due to incorporation of ADP occurs, however, when the proton gradient decreases as a consequence of exhaustion of substrate ATP.

Transient stimulation of light-triggered ATP hydrolysis by preillumination of chloroplasts in the presence of ATP

Abstract Chloroplasts which were preilluminated in the presence of dithiothreitol and ATP + Mg2+ show a 1.5–5-fold higher initial velocity of ATP hydrolysis in the subsequent dark than chloroplasts which were preilluminated in the absence of ATP. The half maximal time of ATP preincubation is about 1 min, the half maximal ATP concentration 15 μM. Nucleotide specificity with regard to the diastereomers of adenosine 5′-(O-1-thiotriphosphate) is the same as in nucleotide binding to CF1 and as the substrate specificity in ATP hydrolysis. ADP abolishes the stimulating effect of ATP if simulaneously present in the light. ATP-stimulated ATP hydrolysis generates a lower transmembrane ΔpH than normal ATP hydrolysis and the relaxation of the flash-induced electrochromic absorption change is accelerated by ATP-preincubation in the light. These results suggest that the ATP pretreatment causes an increase of thylakoid membrane permeability. The target for ATP is most likely the ATPase itself. Dithiothreitol activation in the presence of ATP might induce a reversible dislocation of CF1 relative to CF0 resulting in transitory membrane leakage. Reversibility is demonstrated by the finding that the stimulated rate of ATP hydrolysis is normalized after about 1 min. Moreover ATP P i exchange which exhibits a lag under these conditions recovers up to the control rate after 1 min.

Equilibration of the ATPase reaction of chloroplasts at transition from strong light to weak light

Abstract Broken chloroplasts activated by preillumination in the presence of dithiothreitol were supplied with phosphate and with a limited concentration of ADP. On re-illumination, ATP was formed until a steady state was attained. If after reaching the steady state light intensity was reduced to 20–50 W · m−2, net ATP hydrolysis took place, but after some time in weak light the level of ATP re-increased. Similarly, a drop of transmembrane ΔpH followed by a slow recovery was observed. Further data indicate that the reversible changes of ATP level and ΔpH are the result of partial uncoupling induced by ATP during the preceding strong light period and of restoration of coupling within a few minutes in weak light. Since similar changes of endogenous ATP level were found when intact chloroplasts were subjected to a strong-light/weak-light transition, it is proposed that ATP-induced partial uncoupling may play a role in regulation of photosynthetic energy conservation as a means to dissipate abundant transmembrane electrochemical energy and to permit flexibility of the stoichiometry of ATP-to-NADPH production.

The effect of azide on regulation of the chloroplast H+-ATPase by ADP and phosphate

The light + dithiothreitol-induced chloroplast ATPase is rapidly deactivated by tight binding of ADP to CF 1 whereas ADP binding and ADP-dependent inactivation, respectively, are decelerated by inorganic phosphate. Sodium azide specifically prevents the effect of phosphate on ATPase activity but does not change tight ADP binding significantly. Azide is concluded to interact as a competitor to phosphate with an intermediate enzyme form containing a loosely boundADP. The ATPase · azide · ADP complex, in contrast to the ATPase · phosphate · ADP complex, forms an inactive enzyme species.