Table II.

Effect of calmodulin or peptide 20–44 on the Vmax and Km for Ca2+ of ACA2

ACA ProteinAdditionKm for CaVmax
μm nmol mg−1min−1
Full-length− Calmodulin0.62 ± 0.022.27 ± 0.07
 ACA2-1+ Calmodulin0.43 ± 0.055.10 ± 0.26
Truncated− Peptide 20–440.25 ± 0.027.07 ± 0.62
 ACA2-2+ Peptide 20–440.51 ± 0.033.34 ± 0.22

The effect of 0.5 μm calmodulin on theK m for Ca2+ and theV max of the full-length ACA2-1 were estimated from several Lineweaver-Burk plots. Membranes were isolated from ACA2-1 transformants. EGTA (100 μm) was added to reaction mixtures containing 1 to 100 μm Ca2+ to give free Ca2+ concentrations from 1 nm to 2.6 μm. The initial rate of vanadate-sensitive Ca2+ transport was determined at 30 s in the presence or absence of calmodulin. The effect of peptide 20–44 on45Ca2+ pumping by the truncated ACA2-2 was determined as a function of Ca2+ concentration as described above. Microsomal membranes were incubated with or without 4 μm peptide 20–44 for 10 min. Transport was started with ATP and stopped at 30 s. Pump activity was determined as orthovanadate-inhibited and bafilomycin- and CCCP-resistant Ca2+ uptake. Averages (±se) from three to four experiments using three separate membrane preparations are shown.