Characterization of a Partially Purified Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

Abstract

The (K+,Mg2+)-ATPase was partially purified from a plasma membrane fraction from corn roots [Zea mays L.] (WF9 .times. Mol7) and stored in liquid N2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+) when assayed at 3 mM ATP and 3 mM divalent cation at pH 6.3. When assayed in the presence of 3 mM Mg2+, the enzyme was activated by monovalent cations (K+, NH4+, Rb+ .mchgt. Na+, Cs+, Li+). The pH optima were 6.5 and 6.3 in the absence and presence of 50 mM KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 mM in the absence and 0.7 mM in the presence of 50 mM KCl. Stimulation by K+ approached simple Michaelis-Menten kinetics, with a Km of .apprx. 4 mM KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 .mu.M in the absence and presence of 50 mM KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K+ and preferred Mn2+ to Mg2+. The (K+,Mg2+)-ATPase may be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.