Rapid detergent exchange in solutions of the membrane protein bacteriorhodopsin by preparative high-performance liquid chromatography (HPLC)

Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes. A comparison between the efficiency of monomeric bacteriorhodopsin and purple membrane patches in coreconstitution experiments

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-1 mg ATP synthase-1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-1 mg-1 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 pr…

Time-dependent monomerization of bacteriorhodopsin in triton X-100 solutions analyzed by high-performance liquid chromatography

Abstract Bacteriorhodopsin from Halobacterium halobium was monomerized in Triton X-100 solutions. The process of delipidation was monitored by size-exclusion high-performance liquid chromatography under conditions that preserved the native conformation of the protein. The effects on the process of monomerization of the concentration and pH of the Triton X-100 solutions were investigated. The monomeric bacteriorhodopsin separated was active in light-dependent proton translocation when incorporated into soy bean lecithin liposomes.

Pump and Displacement Currents of Reconstituted ATP Synthase on Black Lipid Membranes

Purified ATP synthase (F0F1) from Rhodospirillum rubrum was reconstituted into asolectine liposomes which were then adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with u.v.-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 (Pi). In analogy oligomycin sensitive pump currents in the reverse direction coupled to n…