Two functional states of sarcoplasmic reticulum ATPase

Area of Science:

• Biochemistry
• Membrane Protein Function
• Enzyme Kinetics

Background:

• Rabbit sarcoplasmic reticulum (SR) exhibits distinct Ca2+-independent ("basic") and Ca2+-dependent ("extra") ATPase activities.
• Only the Ca2+-dependent ATPase activity is coupled to calcium ion (Ca2+) transport.
• SR preparations can be fractionated, revealing variations in the ratio of these activities.

Purpose of the Study:

• To investigate the functional states of rabbit sarcoplasmic reticulum (SR) ATPase.
• To elucidate the relationship between ATPase activity, Ca2+ transport, and membrane environment.
• To understand the temperature-dependent modulation of SR ATPase activity.

Main Methods:

• Fractionation of SR vesicles using density-gradient centrifugation.
• Measurement of "total", "basic", and "extra" ATPase activities at varying temperatures.
• Analysis of steady-state levels of phosphorylated SR protein.
• Effect of Triton X-100 on ATPase activity and substrate dependence.

Main Results:

• The ratio of "extra" to "basic" ATPase activity is temperature-dependent.
• Steady-state phosphorylation levels remain similar across fractions and temperatures.
• Triton X-100 induces Ca2+ dependence in "basic" ATPase and simplifies substrate kinetics.
• SR ATPase exists in two functional states (E1 and E2), with E2 coupled to Ca2+ transport.

Conclusions:

• The E1 <=> E2 equilibrium is temperature-dependent, entropy-driven, and linked to the protein's membrane environment.
• Non-linear Arrhenius plots reflect contributions from both catalytic activation and E1-E2 conversion.
• Membrane structure and viscosity significantly influence the thermal equilibrium between functional states.