Table I.

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

FractionProteinCa2+-ATPase Activity
TotalSpecific
−CaM+CaM−CaM+CaM
mg nmol Pimin−1 μmol min−1 mg−1protein
PM41913370.050.08
Solubilized PM3.41722550.050.07
CaM-agarose:
 Unbound2.3961030.040.04
 I WashND2631
 II WashND55
 1 mm EGTA, I eluateND79
 1 mm EGTA, II eluateND55
 5 mm EDTA eluate0.0524440.430.82

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 the “Materials and Methods.” The first wash was performed in the presence of 100 μmCaCl2 and 100 μm MgSO4; the second one in the absence of added divalent cations. Ca2+-ATPase activity was measured as Ca2+-dependent ITPase activity plus or minus 20 μg mL−1 CaM; ITPase activity measured in the absence of Ca2+ was about 100 nmol Pi min−1in the native and solubilized PM and in the fraction which did not bind to CaM-agarose and barely detectable (1–3 nmol Pimin−1) in the EDTA eluted fraction. Results are from one experiment, representative of more than 10. ND, Not determined.