Nucleotide-binding properties of native and cold-treated mitochondrial ATPase.

Area of Science:

• Biochemistry
• Mitochondrial Function
• Enzyme Kinetics

Background:

• Beef-heart mitochondrial ATPase (F1) is crucial for cellular energy production.
• Understanding its stability and nucleotide interactions is key to elucidating energy transduction mechanisms.

Purpose of the Study:

• To investigate the effects of cold inactivation and ammonium sulfate precipitation on mitochondrial ATPase (F1) activity and nucleotide binding.
• To explore the mechanisms of ATPase reactivation and the role of adenine nucleotides.

Main Methods:

• Cold inactivation of beef-heart mitochondrial ATPase (F1).
• Ammonium sulfate precipitation and assessment of sedimentation coefficients (s20, w).
• Nucleotide exchange assays during enzyme reactivation.
• Enzyme activity measurements under various conditions (glycerol, ATP, ADP, GTP, CTP, pyrophosphate).

Main Results:

• Cold inactivation and precipitation lead to loss of bound nucleotides and ATPase activity.
• ATPase dissociates into smaller subunits (9 S and 3.5 S) during inactivation and precipitation, with nucleotide loss occurring upon further dissociation.
• Reactivation at 30°C is enhanced by glycerol but inhibited by ADP, indicating nucleotide exchange.
• Cold-inactivated ATPase exhibits more extensive and faster nucleotide exchange than the native enzyme.

Conclusions:

• Cold inactivation and ammonium sulfate precipitation destabilize mitochondrial ATPase (F1), causing nucleotide loss and dissociation.
• ADP inhibits reactivation by exchanging with bound ATP, forming an inactive complex.
• Glycerol facilitates reactivation, potentially by stabilizing the enzyme structure during nucleotide exchange.