Table II.

Effect of calmodulin or peptide 20–44 on the V_max and K_m for Ca²⁺ of ACA2

ACA Protein	Addition	Km for Ca	Vmax
		μm	nmol mg−1min−1
Full-length	− Calmodulin	0.62 ± 0.02	2.27 ± 0.07
ACA2-1	+ Calmodulin	0.43 ± 0.05	5.10 ± 0.26
Truncated	− Peptide 20–44	0.25 ± 0.02	7.07 ± 0.62
ACA2-2	+ Peptide 20–44	0.51 ± 0.03	3.34 ± 0.22

The effect of 0.5 μm calmodulin on the K_m for Ca²⁺ and the V_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 on ⁴⁵Ca²⁺ 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.