Table I.

Purification of the PM Ca2+-ATPase by CaM-agarose affinity chromatography

FractionProteinCa2+-ATPase Activity
TotalSpecific
−CaM+CaMΔCaM−CaM+CaMΔCaM
mgnmol Pi min−1 μmol min−1 mg−1 protein
PM5.796260164  (+171%)0.0170.0450.028
Solubilized PM4.190200110  (+122%)0.0220.0490.027
CaM-agarose
 Unbound3.6809010  (+12%)0.0220.0250.003
 Wash no. 1ND-a 11121  (+9%)
 Wash no. 2ND44
 1 mm EGTA, eluate no. 1ND9112  (+22%)
 1 mm EGTA, eluate no. 2ND9123  (+33%)
 5 mm EDTA eluate0.02165842  (+260%)0.8 (×47)2.9 (×65)2.1 (×75)

PM proteins were solubilized with n -dodecyl β-d-maltoside (4:4 mg detergent mL−1: mg protein mL−1) and purified by CaM-agarose-affinity chromatography as described in Methods. The first wash was performed in the presence of 100 μm CaCl2 and 100 μm MgSO4; the second one was performed in the absence of added divalent cations. Data in the brackets represent the percent stimulation by CaM (20 μg mL−1). Results are from one experiment, which is representative of more than 10 experiments.

    • F0-a ND, Not determined.