Steady state kinetics of ATP synthesis and hydrolysis coupled calcium transport catalyzed by the reconstituted sarcoplasmic reticulum ATPase.
The steady state kinetics of calcium transport driven by ATP hydrolysis and ATP synthesis catalyzed by purified, reconstituted calcium ATPase has been investigated as a function of the transmembrane calcium gradient. Purified calcium ATPase was reconstituted into phospholipid vesicles enabling control of the transmembrane calcium gradient. Calcium transport was monitored spectrophotometrically by the calcium indicator, Arsenazo III. Thus, only the enzymatic activity of coupled transport was measured. It was shown under conditions of low external calcium that ATP hydrolysis and synthesis follow simple Michaelis-Menten kinetics and that Michaelis constants obtained for both processes appear independent of the calcium gradient. The maximum velocities for both hydrolysis and synthesis strongly depend on the transmembrane calcium gradient. Based on these results, a mechanism is proposed in which a random addition of substrates for ATP synthesis is followed by random release of ATP and calcium. By measuring the ATP hydrolysis and synthesis under identical conditions, determination of the equilibrium constant for ATP hydrolysis as a function of the transmembrane calcium gradient was possible. Our results indicate that the thermodynamics of the catalytic cycle can be totally accounted for by the energetics of transport of 2.2 +/- 0.3 calciums and the hydrolysis of 1 ATP. An equilibrium constant for ATP hydrolysis in the absence of a calcium gradient was determined to be 4.0 X 10(4).
Fagan
M H
MH
Dewey
T G
TG
D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013816:Thermodynamics
D008081:Liposomes; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium