Acta Botanica Sinica Volume 39 Issue 4, Pages .
Proton-Conductivity of CF0-CF1 Reconstructed into Planar Lipid Bilayer

Wei Jin, He Xiang-dong, Ma Xue-hai and Shi Yu-liang

Abstract:
The proton conductable ATP synthase (CF0-CF1) is the key enzyme of energy conversion in the membrane of bacteria, mitochondria and chloroplast. In spite of a large body of studies, the structure and molecular mechanism of ATP synthases are still elusive. In order to learn the mechanism of ATP synthases, the authors used voltage-olamp technique to study the effect of different conditions on the proton conductance of F0-F1 into planar lipid bilayer membrane. The results obtained were as follows: (1) When CF0-CF1 was reconstructed into planar lipid bilayer membrane, the resistance decreased by 10 times. (2) Channel-like current was recorded at the low concentration of CF0-CFl(protein 2 mg/L) in the solution. (3) In metal ion-free solution, the channel currents changed with the trans-membrane proton gradient (ApH). Under holding potential from 0 to + 150 mV, the stimulation of ¡÷pH on channel current increased with a rise in the ApH from 2 to 4, the stimulation of 4.5 ¡÷pH on channel current was weaker than that of ¡÷pH 4.0. (4) The proton conduetance inhibitor, dicyclohexylcarbodiimide (DCCD), showed a rapid and irreversible inhibition effect on the channel current. (5) In metal ion-free solution (10 mmol/L Tris-HC1), when the ApH across the black lipid membrane (BLM) maintained at 3.0, the addition of Mg2 + caused a alger channel current of CF0-CF1 than the addition of Ca2+ , with holding potential from 0 to + 150 mV. The results indicated that reconstruction of CF0-CF1 was successful and Mg2 + was directly involved in the proton conductance pathways.

Keywords:
H+-ATPase complex, Proton conduction, Voltage-clamp, Planar lipid bilayer
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