Extraction and purification of the beta subunit and an active alpha beta-core complex from the spinach chloroplast CFoF1-ATP synthase.
Incubation of Rhodospirillum rubrum chromatophores with 2 M LiCl in the presence of MgATP has been shown to remove their F1 beta subunit leaving inactive but fully reconstitutable beta-less chromatophores (Gromet-Elhanan, Z., and Khanashvili, D., (1986) Methods Enzymol, 126, 528-538). A similar treatment of thoroughly washed spinach thylakoids has now been shown to release the CF1 beta subunit (CF1 beta) together with a complex containing equal amounts of CF1 alpha and CF1 beta (CF1 (alpha beta]. The purified CF1 (alpha beta) complex can reconstitute an active membrane-bound hybrid F0F1-ATPase with beta-less R. rubrum chromatophores and also catalyzes a low but very reproducible soluble MgATPase. Purified CF1 beta shows none of these activities although it can bind as efficiently as CF1 (alpha beta) to the beta-less chromatophores. By subjecting the crude spinach 2 m LiCl extract to dissociating conditions an enriched CF1 beta preparation is released. It contains traces of CF1 alpha and CF1 delta, is able to reconstitute an active hybrid F0F1-ATPase but, as the pure CF1 beta shows no soluble ATPase activity. These results indicate that trace amounts of CF1 alpha are enough for endowing CF1 beta with a reconstitutive capacity, but for exhibition of a significant soluble ATPase activity equivalent amounts of CF1 alpha and beta are required. The CF 1 (alpha beta) complex isolated and purified in this report thus represents the minimal catalytic core of the CF1-ATPase.
Avital
S
S
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Gromet-Elhanan
Z
Z
D015153:Blotting, Western; D002736:Chloroplasts / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D010785:Photophosphorylation; D010944:Plants / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases