Table I.

Ca2+ pump is detected after blocking H+/Ca2+ antiport activity

Transformant MembraneCa2+ Uptake
− Oxalate+ Oxalate
TotalPumpAntiportTotalPumpAntiport
nmol/mg protein at 15 min
Control1.320.071.253.410.103.31
Full-length ACA2-10.240.220.020.720.450.27
N-Truncated ACA2-20.990.880.117.005.931.07

Membranes (20/45%) were isolated from yeast mutant K616 transformed with vector alone (control), ACA2-1 (encoding full-length protein), or ACA2-2 (encoding N-truncated protein). ATP-dependent 45Ca2+ uptake at 15 min was determined in a 250-μL reaction mixture containing 250 mmSuc, 25 mm HEPES-BTP (pH 7.0), 10 mm KCl, 3 mm MgSO4, 3 mm ATP, 0.2 mm NaN3, 10 μm CaCl2(0.25 μCi/mL), and 100 μm EGTA to give a final [Ca2+] of 50 nm. ΔpH-dependent activity (antiport) was estimated as the difference between Ca2+uptake in the absence of any inhibitor or ionophore (total) and that in the presence of 1 μm bafilomycin A1 and 5 μm CCCP (pump). Uptake with 10 mm K-oxalate was linear for at least 30 min.