Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl₂ and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.     

Kinetics of the H-ATPase in chromatophores from Rhodospirillum rubrum: Effect of the electrical transmembrane potential on the rate constants

The effect of the electrical potential on the H⁺-ATPase of Rhodospirillum rubrum is examined. It is shown that the forward reaction rate (ATP synthesis) is increased by a factor of 10 during illumination while the reversed rate is only slightly decreased. This indicates that the electrical potential across the membrane affects the rate constants mainly by increasing the forward rate constants rather than decreasing the reversed rate constants in order to go from net hydrolysis to net synthesis.     

Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and F0-F1 complex from Rhodospirillum rubrum

PPi driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PPiase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze-thaw procedure. In the presence of ADP, Mg2+, Pi and PPi the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein-1 X min-1, at 20 degrees C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N'-dicyclohexylcarbodiimide.     

Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores

Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing P(i) as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg(2+) concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores.     

Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures

Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum.     

Energy-linked reactions in photosynthetic bacteria: Pi in equilibrium with HOH oxygen exchange catalyzed by the membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum

The inorganic phosphate-water oxygen exchange reaction has been studied in chromatophores of Rhodospirillum rubrum. Under appropriate conditions, chromatophores catalyzed this exchange at a rate of up to 150 μatom oxygen/h/mg bacteriochlorophyll. The reaction is largely inhibited by inhibitors of the membrane-bound inorganic pyrophosphatase, fluoride and methylene diphosphonate, and is not inhibited by oligomycin. These results indicate that the P_i ? HOH oxygen exchange is almost entirely due to the pyrophosphatase. In the presence of ADP, the exchange reaction was stimulated by about 40% and this portion of the exchange was sensitive to oligomycin, but not to fluoride or methylene diphosphonate. Thus this portion of the exchange can be attributed to the ATP synthese complex. The rates of the oxygen exchange reaction and other chromatophore-catyalyzed reactions are compared.     

Reconstruction of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Using the freezing-thawing procedure, a highly purified preparation of PPase from R. rubrum chromatophore membranes was incorporated into soybean phospholipid liposomes. The activity of reconstituted PPase was increased in the presence of the uncoupler, FCCP, and the antibiotics, valinomycin (+KCl) and nigericin (+KCl). Oligomycin did not exert any inhibiting action, while imidodiphosphate and NaF significantly decreased the activity of the PPase incorporated into the liposomes. Preincubation of both PPase and ATPase prior to their incorporation into the liposomes did not affect the activity of the reconstituted enzyme. It was concluded that the PPase from R. rubrum chromatophores when incorporated into the liposomes may function as a proton pump independently of the ATPase.     

Reconstitution of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Overt carnitine palmitoyl transferase (CPT₁) activity was measured in liver mitochondria from foetal rats (21 days gestation) and from neonatal rats (1 day post-partum). Birth was accompanied by a 6-fold increase in CPT₁ activity, a 14-fold decrease in sensitivity to inhibition by malonyl CoA and an increase in the n_H and the S_0.5 from palmitoyl CoA. The activity of latent enzyme (CPT₂) was unaffected at birth.     

Size distribution and photophosphorylation of chromatophores from t Rhodospirillum rubrum

Morphology and photophosphorylation of chromatophores from t Rhodospirillum rubrum have been investigated by dynamic light scattering (DLS) and in situ ³¹P-NMR measurement. Two components, designated as light and heavy fractions, with different average sizes and size distributions were detected by the DLS and can be separated by sucrose density gradient centrifugation. The light fraction has an average size of about 140 nm in diameter with a narrow distribution and shows a high activity of photophosphorylation. About 70 of ADP were found to be converted to ATP purely by the photophosphorylative reaction. In contrast, the heavy fraction has a broad size distribution centered around 350 nm and a low activity of photophosphorylation. Only about 50 of ADP was converted into ATP and AMP with a ratio of 7:3, indicating that most membrane-bound adenylate kinase are attached on the particles of the heavy fraction. Effect of physical disruption on the structural integrity of chromatophores has been examined by using sonication with various oscillating strengths. The result shows that the morphology of chromatophores for both light and heavy fractions is relatively stable to the disruption, while the photophosphorylative activity of the light fraction is very sensitive to the disrupting strength, suggesting that the internal structure of the purified chromatophores could be partially damaged by the disruption.     

Nanosecond fluorescence from chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.