Chanoch Carmeli

Effects of Pi and ADP on ATPase activity in chloroplasts

Abstract 1. 1. The rate of decay of the light-triggered state of ATPase in chloroplasts was decreased in the presence of Pi and accelerated by ADP in a phosphateless medium. Phosphate and arsenate inhibited the effect of ADP. 2. 2. Compared with other dinucleotides, ADP was found to be highly specific for the acceleration of the decay, having an apparent Km of 1.1·10−6 M. A lower apparent Km for ADP was obtained in the presence of phosphate. The apparent Km for the inhibition of the effect of ADP by Pi was lowered with increased concentrations of ADP. 3. 3. These and other effects on ATPase and ATP-Pi exchange activities were discussed in terms of changes in the permeability of ADP and Pi across the membrane of the chloroplast.

Proton translocation induced by ATPase activity in chloroplasts.

Proton uptake by chloroplasts was induced by light‐triggered ATPase activity. A quotient of two was obtained when the initial rate of proton uptake was divided by the rate of Pi released from ATP. Gramicidin accelerated the rate of ATPase activity and reduced the H+/Pi ratio to 1.4. The results were found to be consistent with the chemiosmotic theory.

Relations between divalent cation binding and ATPase activity in coupling factor from chloroplast

Coupling factor from chloroplast (CF,) was found to be directly involved in the terminal step of photophosphorylation. CFr also catalyzes ATP hydrolysis which is probably a result of the reversal of ATP synthesis. However, this enzymatic activity is latent whether the protein is bound to the chloroplast membrane or isolated in a purified soluble form. Several methods for the unmasking of ATPase activity were devised [l-4]. The catalysis of ATP hydrolysis by activated CFr was found to be dependent on divalent cations [2,5]. Based on kinetic analysis of ATPase activity in photosynthetic bacterial chromatophores we have suggested that the cation-ATP complex is the true substrate for ATPase activity [6]. This kinetic work is extended here in order to probe the mode of action of ATPase in purified CFr. The kinetic data indicated that the cation-ATP complex was also substrate for ATPase activity in CFr . Based on the analysis of inhibition it is suggested that the complex is attached at least at two points to the enzyme. One point of attachment is through the cation in the complex and the other through part of the ATP molecule. The measurements of cation binding were of great relevance to the understanding of the mode of action. Direct binding of Mn*+ions as measured by the e.p.r. method indicated two tight and three loose cation binding sites. Based on the interpretation of the kinetic and binding data, suggestions were made as for the possible mode of substrate and inhibitors binding to the active site. The results were compared to measurements of nucleotide binding to CFr [7-91.

Properties of ATPase in chloroplasts

Abstract 1. 1. Mg 2+ -ATPase (light-triggered Mg 2+ -dependent ATPase) activity in chloroplasts was stimulated by atebrin, NH 4 Cl and gramicidin when the uncouplers were added after light triggering. The stimulation was followed in time by inhibition when the reaction took place in the dark. 2. 2. This inhibition of Mg 2+ -ATPase activity was overcome when the reaction was carried out under continuous illumination. 3. 3. The energy transfer inhibitors of photophosphorylation, phlorizin and Dio-9, inhibited Mg 2+ -ATPase activity and the extent of inhibition increased with time. The inhibition by Dio-9 was partially reversed by light while that of phlorizin was not. 4. 4. Light-triggered ATP-P i exchange activity in chloroplasts was inhibited by both atebrin and phlorizin. The extent of the inhibition increased with time. 5. 5. The activity of a soluble Ca 2+ -ATPase was inhibited by Dio-9, phlorizin, NH 4 Cl and atebrin. The kinetics of activity was linear with time, except in the presence of phlorizin. 6. 6. These results are interpreted as indicating a requirement of a high-energy state for triggering and maintenance of Mg 2+ -ATPase and ATP-P i exchange reactions. The relation of ATPase activity to the coupling mechanism in chloroplasts is discussed.

Large Photovoltages Generated by Plant Photosystem I Crystals

Light energy conversion to electrical energy in photosynthesis and in photovoltaic devices is initiated by separation of charges that generates a photovoltage across an energy barrier in the system. [ 1–3 ] In conventional devices, mostly based on semiconductors, [ 4 ] the photovoltage is limited by the bandgap energy. Anomalous photovoltaic effects, in which the generated photovoltage is larger than the bandgap, have been reported in polar materials such as ferroelectric crystals, [ 5 , 6 ] but also in various amorphous and polycrystalline semiconductors. [ 7–10 ] Can a photoactive biological protein also generate an anomalous photovoltage? Natural candidates are photosynthetic reaction centers such as plant photosystem I (PSI), which are highly effi cient light energy converters found in thylakoid membranes inside the chloroplasts of green plants. [ 11 , 12 ] Here we show that these dried micrometer-thick crystalline biological protein complexes made of plant PSI [ 13 ] can generate unprecedented high photovoltages (well above 10 V) when placed on a conducting solid surface and measured by Kelvin probe force microscopy (KPFM). The measured photovoltages give rise to internal electric fi elds as large as 100 kV cm − 1 , which are among the highest values ever reported in an inorganic material system. We suggest that these high values result from depletion of the P700 electron donor centers and trapping centers in the PSI complex. We also present an equation depicting the dependence of the large photovoltage phenomenon in plant PSI crystals on light intensity. The unprecedented photovoltage, the microscale dimensions, and the high quantum yield make the PSI crystals intriguing units for potential use in optoelectronic devices. Photosynthetic reaction centers such as PSI evolved over a billion years to become highly effi cient light energy converters.

Control of proton translocation induced by ATPase activity in chloroplasts.

1. Proton uptake was induced by ATP in the dark following light triggering of ATPase activity in chloroplasts. The accumulated protons were released when ATPase activity was inhibited by the energy transfer inhibitor DIO-9. 2. Approximately two protons were taken up for each ATP hydrolyzed at pH 8. A drop in H+/ATP ratio was caused by uncouplers such as NH4Cl and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These uncouplers caused an increase in the rate of ATP hydrolysis without a corresponding increase in proton uptake. 3. The energy transfer inhibitor dicyclocarbodiimide inhibited both ATPase activity and the rate of proton uptake without changing the H+/ATP ratio. 4. The antibiotic valinomycin caused an increase in the rate of both proton uptake and ATP hydrolysis without altering the ratio of H+/ATP. The H+/ATP ratio varied with changes in the external pH. The results were discussed in view of the chemiosmotic theory of oxidative and photosynthetic phosphorylation.

A coupling factor from chromatium strain D chromatophores

The mechanism of photosynthesis in the obligatory anaerobic bacteria chromatium strain D differs in many respects from that of higher plants [l] . However, the bacterial chromatophores resemble chloroplasts in their lack of capacity for oxidative phosphorylation. It was therefore of interest to conduct a comparative study of photophosphorylation in chromatium strain D chromatophores. We attempted to study the partial reactions of photophosphorylation and to isolate a coupling factor. While this work was in progress [2] Baccarini-Melandri and Gest [3] reported a coupling factor from Rhodopseudomonas capsulata. In chloroplasts, the work on coupling factors is much more advanced than in chromatophores. Treatment of chloroplasts with EDTA in a low salt medium released a coupling factor [4]. The same factor could also be isolated from acetone powder of chloroplasts [5]. The coupling factor restored phosphorylation in EDTA washed chloroplasts [4,5] and in nonphosphorylating subchloroplasts particles [5]. Both the chloroplasts and the coupling factor had a latent ATPase activity which could be activated by various treatments [5,7]. An antiserum to the coupling factor inhibited phosphorylation and ATPase activity in the chloroplasts as well as ATPase activity in the isolated coupling factor [8]. These data indicate that the coupling factor is an enzyme which takes part in the terminal step of ATP synthesis in chloroplasts.

Picosecond electron transfer from photosynthetic reaction center protein to GaAs.

An extremely fast electron transfer through an electronically coupled junction between covalently bound oriented photosynthetic reaction center protein photosystem I (PS I) and n-GaAs was measured by time-resolved photoluminescence. It was found that the n-GaAs band edge luminescence intensity increased by a factor of 2, and the fast exponential decay constant was increased by a factor of 2.6 following the PS I self-assembly. We attribute this to picosecond electron transfer from the PS I to the n-GaAs surface states.

Purification and properties of adenosine triphosphatase from Chromatium vinosum chromatophores

washed in 0.1 M tricine-NaOH, pH 7.8 and stored under nitrogen at -2O’C. The cells were ground with alumina in 0.1 M tricine-NaOH, pH 7.8, then centrifuged at 12 000 X g for 15 min to remove debris. The chromatophores were sedimented by cen- trifugation at 144 000 X g for 1 h, resuspended in a small volume of a solution containing 0.1 M tricine-

Control of kinetic changes in ATPase activity of soluble coupling factor 1 from chloroplasts

Coupling factor 1 (CFI) from chloroplasts is directly involved in the terminal steps of photophosphorylation. The enzyme can also catalyze ATPase activity which is probably a result of the reversal of ATP synthesis. However, ATPase activity is latent both in the membrane bound and in the isolated soluble pro