Correlation between Ca2+ uptake, Ca2+ efflux and phosphoenzyme level in sarcoplasmic-reticulum vesicles

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Kinetics

Background:

• Dimethyl sulfoxide (Me2SO) was previously shown to increase Ca2+ accumulation by sarcoplasmic reticulum vesicles.
• This effect was linked to a decrease in enzyme affinity for ADP, from <20 μM to 1 mM.
• Understanding ADP's role in enzyme regulation is crucial for sarcoplasmic reticulum function.

Purpose of the Study:

• To determine the apparent affinity of the ADP-sensitive phosphoenzyme for ADP under various conditions.
• To investigate the influence of Me2SO and KCl on ATP synthesis and Ca2+ accumulation.
• To elucidate the mechanism by which ADP binding affects enzyme conformation and Ca2+ transport.

Main Methods:

• Measurement of ATP synthesis rates in Ca2+-loaded sarcoplasmic reticulum vesicles.
• Determination of ADP affinity (Km) at different pH values, with and without Me2SO and KCl.
• Analysis of enzyme phosphorylation by inorganic phosphate (Pi) and ADP inhibition kinetics.

Main Results:

• Me2SO did not increase the apparent Km for ADP beyond 25 μM under tested conditions.
• ADP inhibition of Pi phosphorylation showed two components (Ki: 80 μM and 8 mM); Me2SO abolished the high-affinity component.
• Me2SO decreased passive Ca2+ efflux and increased the total phosphoenzyme fraction during Ca2+ uptake.

Conclusions:

• ADP binds to at least two distinct enzyme conformations, one of which (*E) inhibits Pi phosphorylation and increases Ca2+ efflux.
• Me2SO alters enzyme conformation, reducing high-affinity ADP binding and enhancing Ca2+ storage.
• These findings provide insights into the regulation of Ca2+ transport by sarcoplasmic reticulum ATPases.