Rapid Purification of Plant Plasma Membrane K+,Mg2+-ATPase on a Phosphocellulose Column

Plasma membrane K⁺,Mg²⁺-ATPase with high specific acitivity was purified from soybean (Glycine max L. cv. Williams) hypocotyls. Aqueous polymer two-phase partition-purified PM vesicles were solubilized by the non-ionic detergent, C₁₂E₈, and P-11 phosphocellulose fibers were used to bind the solubilized ATPase. ATP hydrolyzing enzymes bound to the phosphocellulose fibers were eluted at 0.6 M NaCl during the step gradient elution. The pH dependence of the Mg activation, as well as that of its K⁺ stimulation and orthovanadate inhibition of the desalted and concentrated ATP-hydrolyzing fractions was identical to those obtained with the purified PM vesicles. The specific activity, however, was about 20 times higher in the fractions containing the partially purified enzyme than with the phase partition purified PM vesicles. This value was proportional to the protein yield of purification.