Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.
The finding that negatively charged phospholipids activate the plasma-membrane (Ca2+ + Mg2+)-ATPase and that polycations counteract this stimulation suggest that negative charges in the environment of the ATPase protein could be important for its function. The aim of the present work was to investigate whether changing the charges on the ATPase protein itself by modifying the pH within the physiological range affects the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle. Increasing the pH from 6.9 to 7.4 and using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) as a Ca2+ buffer, doubled the ATPase activity at 0.3 microM-Ca2+ in the presence of 100% phosphatidylcholine (PC) or after substituting 20% of the PC by negatively charged phospholipids PtdIns, PtdIns4P, phosphatidylserine and phosphatidic acid. This stimulatory effect was due to an increased affinity of the enzyme for Ca2+, while the Vmax. remained unaffected. In the case of PtdIns(4,5)P2, a stimulatory effect upon alkalinization was only observed at a PtdIns(4,5)P2 concentration of 10%. When a concentration of 20% was used, alkalinization decreased the Vmax. and no stimulatory effect on the ATPase at 0.3 microM-Ca2+ could be observed. Alkalinization not only stimulated the purified Ca2+ pump, but it also increased the activity of the enzyme in a plasma-membrane-enriched fraction from stomach smooth muscle by a factor of 2.06. The ionophore A23187-induced Ca2+ uptake in closed inside-out vesicles also increased by a factor of 2.54 if the pH was changed from 6.9 to 7.4. This finding indicates that the effect of pH is most likely to be exerted at the cytoplasmic site of the Ca2+ pump protein.
Missiaen
L
L
Physiological Laboratory, K. U. Leuven, Campus Gasthuisberg, Belgium.
Droogmans
G
G
De Smedt
H
H
Wuytack
F
F
Raeymaekers
L
L
Casteels
R
R
D000818:Animals; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D013552:Swine
D010713:Phosphatidylcholines; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium