Catalysis and thermostability of mitochondrial F1-ATPase in toluene-phospholipid-low-water systems.
Soluble mitochondrial F1-ATPase from bovine heart can be transferred to systems composed of a nonpolar solvent (toluene), phospholipid, and water at concentrations between 0.02 and 0.05% (volume of water per volume toluene). In these systems, F1 becomes resistant to cold denaturation and acquires a remarkable thermostability; i.e., its half-life at 70 degrees C is more than 24 h. Thermostability is due to the low content of water, since increases of water concentration bring about a progressive decrease in thermostability. At 0.04% water, the enzyme fails to catalyze a single splitting of ATP per enzyme. Gradual increases in water concentration up to 2.5% result in a progressive increase of hydrolytic activity. However, even at 2.5% water, the activity is orders of magnitude lower than in totally aqueous media. At various concentrations of water (0.1-2.5% v/v) and Mg-ATP, it was found that water affects the Vmax, but not the Km. The results show that, at levels of water below 0.04% (v/v), the enzyme is in a state that does not carry out catalysis and possesses high thermostability. As the water content is increased, the enzyme acquires the progressive flexibility that is required for catalysis and for undergoing rapid thermal denaturation.
Garza-Ramos
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Departamento de Bioquímica, Instituto Politécnico Nacional, México, D.F.
Darszon
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A
Tuena de Gómez-Puyou
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M
Gómez-Puyou
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A
D000818:Animals; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D010743:Phospholipids; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013696:Temperature; D014050:Toluene; D014867:Water
D010743:Phospholipids; D014867:Water; D014050:Toluene; D006180:Proton-Translocating ATPases