The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-' mg ATP synthase-was acheved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-' mg-' found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches.The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution procedure. Furthermore, the ratio (lipid to bacteriorhodopsin to ATP synthase) could be optimized. Light-driven ATP synthesis depends also on the type of detergent used. The best result was obtained by deoxycholate. Also the relationship between proton translocation (by bacteriorhodopsin) and ATP synthesis activity was measured. A constant H+/ATP ratio was found at higher light intensities. This ratio increased strongly at lower light intensities.According to the chemiosmotic theory [l] ATP synthesis in chloroplasts, bacteria and mitochondria, catalyzed by ATP synthase, is driven by an electrochemical gradient of protons across the membrane. Quantitative investigations of the mechanisms of ATP synthesis in vivo are problematical owing to the complex structure of the natural systems. A way to overcome some difficulties is the coreconstitution of an isolated proton pump and a purified ATP synthase. In principle the function of such systems has been demonstrated [2-lo]. The proton pump commonly used in these investigations was bacteriorhodopsin in the form of purple membrane patches isolated from Halobacterium halobium. The most effective coreconstitution system yields ATP synthesis rates up to 10% of the maximal in vivo rate when yeast mitochondria1 ATP synthase is used [4, 81. Unfortunately the reported rates for liposomes with incorporated bacterial ATP synthases are less than 1 YO of the in vivo rate [3, 51. This might be due to the instability of the delipidated enzyme and/or the heterogeneous distribution of the ATP synthase and the bacteriorhodopsin among the liposomes. The ATP synthase complex from Rhodospirillum rubrum has been chosen because of the higher stability of the FoFl complex compared with that of other bacterial ATP synthases (Escherichia coli, Micrococcus luteus). In this paper we show that monomeric bacteriorhodopsin, delipidated by a HPLC procedure [l 1 -131, is superior to purple membrane patches in coreconstitution experiments. Various coreconstitution procedures have been optimized. The protein distribution among the liposomes has been analyzed by density gradient centrifugation. MATERIALS AND METHODSThe detergents deoxycholate, taurodeoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio]-propane sulphonate (Chaps) were purchased from Sigma (Miinchen). Luciferin, luciferase, ATP, and ADP (K salt) were product...
Purified ATP synthase (FoFI) from Rhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P~). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and Pi were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2/xsec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 b~sec were obtained. The kinetics of the charge movements were slowed down by F0 specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding to F~ is prevented by the catalytic site-blocking agent NBD-CI. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.