The expression plasmids for the subunit γ (γc) and the subunit ɛ (ɛc) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant γc was mixed with recombinant α and β subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (α3β3γc) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCI2, which induces formation of a disulfide bond between two cysteine residues in γc. Hence, this complex has similar modulation characteristics as CF1.
The effects of recombinant ɛc, and ɛ subunit from TF1 (ɛ1) on α3β3γc were also investigated. ɛc, strongly inhibited the ATP hydrolysis activity of chimeric α3β3γc complex but ɛ1 did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of ɛc, or ɛ1, to the α3β3γt complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity.
These results suggest that (a) the specific regulatory function of γc can be transferred to the bacterial subunit complex; (b) the interaction between the γc subunit and ɛc, strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α3β3 core.
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chloroplast coupling factor 1
- γc and ɛc
recombinant γ and ɛ subunits of CF1 from spinach
F1 from the plasma membrane of thermophilic Bacillus PS3
- γ1, and ɛ2
recombinant γ and ɛ subunits of TF1
F1 from bovine heart mitochondria
F1 from Escherichia coli plasma membranes
the transmembrane electrochemical potential differences of protons
H+-transporting ATP synthase (EC 188.8.131.52)