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Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety
P L Pedersen1, N Williams, J Hullihen
1Laboratory for Molecular and Cellular Bioenergetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Magnesium ions (Mg2+) inactivate rat liver F1-ATPase by causing subunit dissociation. Rebinding F1 to F0 fully protects ATP synthase activity against this Mg2+-induced inactivation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- ATP synthase is a crucial enzyme complex responsible for cellular energy production.
- The F1 moiety of ATP synthase possesses ATPase activity, which is essential for its function.
- Understanding the regulation and stability of F1-ATPase is vital for comprehending energy metabolism.
Purpose of the Study:
- To investigate the mechanism of inactivation of rat liver F1-ATPase by magnesium ions (Mg2+).
- To identify factors and conditions that protect F1-ATPase activity from Mg2+-induced inactivation.
Main Methods:
- Incubation of purified F1-ATPase with MgCl2 at 25°C.
- Assay of ATPase activity and analysis of subunit dissociation using precipitation and solubility.
- Testing the protective effects of various compounds (ATP, ADP, KPi, chelators) and F0 binding.
Main Results:
- Mg2+ inactivates F1-ATPase at concentrations significantly higher than those used in catalysis.
- Inactivation involves the dissociation of alpha subunits and part of the gamma subunit from the F1 complex.
- High concentrations of KPi partially protect F1, while rebinding to F0 completely prevents inactivation.
Conclusions:
- Mg2+-induced inactivation of F1-ATPase is a subunit dissociation process.
- The F0 moiety plays a critical role in stabilizing F1-ATPase against divalent cation-induced inactivation.
- These findings provide insights into the regulation and structural integrity of ATP synthase.