Separation of the Mg2+-ATPases from the Ca2+-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm(3)). Activities of NADH cytochrome (Cyt) c reductase, Ca(2+)-ATPase, and Mg(2+)-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca(2+)-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm(3), a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm(3), a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm(3), and of vanadate-inhibited ATPase at 1.16 g/cm(3). The Ca(2+)-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca(2+)-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg(2+), was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species.