All eukaryotic F-type ATPases pump 3-4 H+ out of mitochondria, or into thylakoids of chloroplasts, per ATP hydrolyzed. Bacterial F-type ATPases pump 3-4 H+ and/or Na+ (depending on the system) out of the cell per ATP hydrolyzed. These enzymes also operate in the opposite direction, synthesizing ATP when protons or Na+ flow through the 'ATP synthase' down the proton electrochemical gradient (the 'proton motive force' or pmf) (). V-type ATPases may pump 2-3 H+ per ATP hydrolyzed, and these enzymes cannot catalyze pmf-driven ATP synthesis. It has been proposed that this difference between F-type and V-type ATPases is due to a 'proton slip' that results from an altered structure in the membrane sector of V-type ATPases (). This probably results from duplication (intragenic and/or intergenic) of the proteolipid (c) subunit. Several intact F-type ATPase complexes have been purified from different fungal species and analyzed for their properties and subunit comopositions after solubilization with various detergents ().
The rotary proton- and sodium-translocating ATPases are reversible molecular machines present in all cellular life forms that couple ion movement across membranes with ATP hydrolysis or synthesis. Sequence and structural comparisons of F- and V-type ATPases have revealed homology between their catalytic and membrane subunits, but not between the subunits of the central stalk that connects the catalytic and membrane components. It has been proposed that these ATPases originated from membrane protein translocases which evolved from RNA translocases (). The Na+-pumping ATP synthase of Acetobacterium woodii has an unusual feature: its membrane-embedded rotor is a hybrid made of F and V-like subunits in a stoichiometry of 9:1, apparently not variable with the growth conditions ().
The physiological function of F1 is ATP synthesis.
a, Schematic view of the membrane-embedded F0F1-ATP synthase in which the proton-driven F0 rotates F1 in a clockwise direction for ATP synthesis.
Mechanically driven ATP synthesis by F1-ATPase - …
The protocol below is for membranes, but can be applied to any ATP synthase or F1-ATPase preparation.
membranes are diluted to protein concentration of 0.01-0.02mg/ml in a buffer containing 20mM HEPES, 5mM MgCl2, 100mM KCl, 5mM KCN, 2.5mM phosphopyruvate, 200uM NADH, 0.1mg/ml pyruvate kinase, 0.1mg/ml lactate dehydrogenase, pH 7.5-8.0.
Reaction is started by addition of ATP to desired final concentration (typically 1 mM) and followed by the decrease in NADH absorption at 340nm wavelength. The molar extinction coefficient of NADH at 340nm is 6220M-1.
Other additions (e.g. inhibitors, uncouplers, LDAO, etc.) can be made during reaction course.
When NADH is exausted (A340 drops below 0.1), another addition of NADH to ~200uM final concentrationcan be made to continue the measurement.
Synthesis and V-ATPase Inhibition of Simplified …
Chloroplast ATP synthase and the enzyme from some photosyntheticbacteriahave 2 different, although similar, -typesubunits in the protontranslocating FO portion, namely and, one copy ofeach.
High homology is found for most of the ATP synthase subunits fromdifferentbacteria and chloroplasts.
Mitochondrial enzyme is much more complex; are described at the moment. Some of these subunits have high homology to bacterial andchloroplast counterparts, especially subunits Alpha, Beta and Gamma inthe F1 portion and subunits and in the FOportion. Many subunits are unique for the mitochondrial enzyme (see for details).However, the catalytic and proton translocating "core" of the enzyme isstill highly homological to that of bacterial and chloroplast ATPsynthase. The overall topology of the enzyme is also quite similar.