BIOCHEMICAL STUDIES ON VACUOLAR ATPase OF LEGUMES: TONOPLAST ATPase FROM PEANUT

Abstract

Tonoplast membranes possess a proton pumping ATPase. This enzyme generates proton gradient which acts as the driving force for transporting solutes across the membrane. In this study peanut seedlings were used as the source of tonoplast membranes. ATPase associated tonoplast membrane fractions were isolated from seven-day-old seedlings by differential and 3-step sucrose-gradient centrifugation. The enzyme was solubilized by 5% Triton X-100. Phospholipid was essential for recovery of the tonoplast ATPase activity in presence of Triton X-100. The enzyme was purified through Sepharose CL-6B column by 27-fold over crude microsomal pellet. The purified enzyme was highly unstable compared to membrane associated enzyme. The Mr of ATP was estimated in the range from 400 kDa to 600 kDa. SDS-PAGE resulted in separation of three major polypeptides of 69, 55 and 20 kDa. Minor bands of 37 and 15 kDa were also co-purified. Membrane bound ATPase was found to be sensitive to N03 and exhibited a sharp pH optimum at 8.0. Km and V^ values of the enzyme determined were 0.15 mM and 3.1 /xinol Pi mg"1 protein h"1 respectively. The enzyme activity was inhibited by DTT, fl-mercaptoethanol and EDTA. The enzyme required divalent cations for its activity, Mg2+ being the most effective. The enzyme was stimulated by CI" ions. Monovalent cations, excepting RbCl, caused about two-fold stimulation in ATPase activity. Amongst various nucleoside phosphates, ATP was the most effective substrate. GTP was also partially hydrolysed. Proton uptake by tonoplast vesicles was determined with acridine orange as pH probe. The proton pumping in tonoplast vesicles was inhibited (53%) by addition of 50 mM nitrate. In in vitro experiment plant hormones like IAA, IBA, NAA, GA3 and kinetin, in general. (i) showed inhibitory effect on the enzyme activity. In case of PAA, increase in enzyme activity was significantly higher than other hormones. In contrast, tonoplast ATPase showed either little or no inhibition in presence of ABA. In in vivo, all the hormones except GA3 showed inhibitory effect. In in vitro, effects of phenolic compounds on the enzyme activity were inhibitory excepting cinnamic acid and in presence of cinnamic acid marked increase in enzyme activity had been observed . Chlorogenic acid showed complete inhibition. In in vivo experiments seeds could not germinate in presence of phenolic compounds. However, stunted growth and with inappreciable enzyme activity could be observed in presence of hydroquinone at low concentrations. Ultrastructure of tonoplast enzyme under electron microscope revealed the presence of head and stalk structures attached to the surface of the vesicles. Characteristic cleft was seen on the head piece of the enzyme structure. Head and stalk structures disappeared in presence of KN03. In contrast, when mitochondrial ATPase was compared to that of tonoplast, it was found that the enzyme lacked cleft and the enzyme structure remained associated with the membrane vesicles in presence of N03. Mitochondrial ATPase also differed from the tonoplast ATPase having shorter enzyme structure.