Characterization of a potassium-stimulated ATPase in membrane fraction isolated from roots of grapevine seedlings

Abstract

A microsomal fraction possessing Mg²⁺-dependent and K⁺-stimulated ATPase activity was extracted by differential centrifugation from roots of grape seedlings (Vitis vinifera L. cv. Verduzzo).
Roots yield from grape seeds was stimulated by means of GA3 and further improved by treatments able to control microbial contamination.
The biochemical characteristics of ATPase activity were studied and compared with those previously reported for roots produced by grape woody cuttings.
The presence of choline-Cl, ethanolamine and glycerol-1-P in addition to BSA, EDTA, PVPP and DTT in the homogenizing medium was obligatory in order to record the K⁺-stimulated component of activity.
The enzyme was activated by Mg²⁺, further stimulated by monovalent ions and showed strong preference for ATP as the substrate and optimum pH at 6.5 in the presence of both Mg²⁺ and K⁺. The effect of different monovalent ions followed a sequence similar to that found in cereal roots preparations, but very different with respect to that recorded for preparations from roots of grape woody cuttings.
K⁺-ATPase activity was inhibited by vanadate and DES whereas molybdate and azide had no or scarce effect . ATPase activity showed a simple Michaelis-Menten saturation with increasing ATP: Mg concentration, and a complex pattern of possible negative cooperativity for K⁺ stimulation.
Microsomes fractionated using sucrose density gradient showed enrichment in plasmalemma vesicles at 1.10-1,15 g ml^-1 density.
This parameter differentiates this fraction from similar preparations containing plasmalemma ATPase obtained from roots of various annual plants.