Characterization of ATPase Activity of Free and Immobilized Chromatophore Membrane Vesicles of Rhodobacter sphaeroides

Abstract

The intracytoplasmic membrane of readily vesiculates when cells are lysed. The resulting chromatophore membrane vesicle (CMV) contains the photosynthetic machineries to synthesize ATP by ATPase. The light-dependent ATPase activity of CMV was lowered in the presence of O₂, but the activity increased to the level observed under anaerobic condition when the reaction mixture was supplemented with ascorbic acid (≥0.5 mM). Cell lysis in the presence of biotinyl cap phospholipid (bcp) resulted in the incorporation of bcp into the membrane to form biotinylated CMV (bCMV), which binds to streptavidin resin at a ratio of approximately 24 μg bacteriochlorophyllml resin. The ATPase activity of CMV was not affected by biotinylation, but approximately 30% of the activity was lost by immobilization to resin. Interestingly, the remaining 70% of ATPase activity stayed constant during 7-day storage at 4°C. On the contrary, the ATPase activity of bCMV without immobilization gradually decreased to approximately 40% of the initial level in the same comparison. Thus, the ATPase activity of CMV is sustainable after immobilization, and the immobilized bCMV can be used repeatedly as an ATP generator.

Light-driven ATP production promotes mRNA biosynthesis inside hybrid multi-compartment artificial protocells

“…Preliminary experiments, summarized in Text S4 and Figure S3, lead us fixing optimal conditions for the assay, i.e., ∼6 × 34 . When 2 mM ADP is employed, the ATP production rate almost reaches the maximum (99.5% of Vmax, considering KM,ADP = 10 µM for the ATP synthase 35,36 ). The measured production of 1.2 mM ATP in 3 minutes corresponds to an ATP synthase turnover number of ∼100 s -1 (or higher), in good agreement with the highest reported values (50 s -1 37 ; or 90 s -1 38 ) (see Text S5 for a commentary).…”

Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells

“…The calculated reaction quotient [ATP]/([ADP][Pi]) ≅ 15 M −1 is equivalent with a proton motive force of ∼130 mV (positive inside chromatophores) if we assume three translocated protons per ATP molecule(44,45). When 2 mM ADP is employed, the ATP production rate almost reaches the maximum (99.5% of V max , considering the ATPsyn Michaelis constant for ADP (K M,ADP ) equal to 10 μM)(46,47). The measured production of 1.2 mM ATP in 3 min corresponds to an estimated ATPsyn turnover number of ∼80 s −1 (or higher), in good agreement with the highest reported values [50 s −1 (48) or 90 s −1 (49)] (see SI Appendix, Text S5 and Table…”

Sustainability of in vitro light-dependent NADPH generation by the thylakoid membrane of Synechocystis sp. PCC6803

“…The recombinant β-subunit with a his 6 -tag at its N-terminus was produced in Syn-AtpB (Table 1 ), and TM was obtained from cells via sucrose density gradient ultracentrifugation. In order to detect F 1 -out vesicles, varying amounts of TM, which were determined with Chl a unless stated otherwise, were loaded onto the Ni–NTA column within the binding capacity of the resin for the tag, as previously described [ 14 ]. Unbound free TM was removed by washing, and the Chl a content of resin-bound TM β-his was determined after acetone/methanol extraction.…”

“…The sustainability of chromatophore membrane vesicles derived from the intracytoplasmic membrane of Rhodobacter sphaeroides has been improved by immobilization on streptavidin resin [ 14 ]. However, the use of resins in large quantities is not cost-effective.…”