Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0.
Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].
D002736:Chloroplasts / Q000201:enzymology*; D007122:Immunoelectrophoresis; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D010944:Plants / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D011522:Protons; D013045:Species Specificity
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011522:Protons; D006180:Proton-Translocating ATPases