Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in KI-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO3 or KCI. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.Membranes from maize roots have been shown to contain at least two proton transporting ATPases (6,28). One ofthese pumps is localized on the tonoplast membrane and is similar to other vacuolar type ATPases being inhibited by NEM and nitrate, but insensitive to vanadate (7,28). The other pump is believed to be localized on the plasma membrane and similar to other E1-E2 type ATPase in forming an aspartyl phosphate intermediate, being sensitive to vanadate and utilizing Mg-ATP as substrate (1,4,6,7,25,28). Transport ATPases of the El -E2 type have been shown to exist in at least two different conformational states depending on the ligands bound to the enzyme (1, 25). These conformational states have been deduced by changes in susceptibility to proteolytic degradation ( 19 and references cited therein) and fluorescence of aromatic amino acids within the protein (14,16) and covalently bound probes (15). It has been postulated that the changes in protein structure are essential for ion movement (25), because the conformation of the E 1-E2 type ATPase is affected by binding of transported cation (14).Characterization of proton transport by the vanadate-sensitive pump from maize roots has been slowed because of difficulties in purifying plasma membranes with competent transport activities. Problems in isolating these membranes arise from the abundance of proteases in membrane fractions (8) and the presence of lipolytic activities which affect membrane integrity (3). Recent advances in purification of membranes from roots have allowed isolation of vesicles with vanadate-sensitive proton transport (6, 7, 10). Additionally, several reconstitution protocols have been developed to insert the vanadate-sensitive ATPase into liposomes (3,26).In a recent article (28), a kinetic model for describing proton transport by the tonoplast ATPase was proposed. This model quantifies the overall process of proton transport by simultaneously considering the pumping and the leakage of protons from membra...

Autolytic lipid changes in corn (Zea mays L.) root crude homogenates and isolated membranes were examined by the use of high performance thin-layer chromatography. In the absence of added CaCI2, losses in phosphatidylcholine and other phospholipids corresponds to increase in fatty acids without the accumulation of either phosphatidic acid or lyso-phosphatidylcholine. However, in the presence of 1 millimolar CaCI2, phosphatidylcholine concentrations declined more rapidly with an immediate increase in phoshatidic acid, and slower rate of fatty acid accumulation. Autolytic phospholipid degradation yielded primarily free fatty acids in the absence of Ca and phosphatidic acid in the presence of 1 millimolar CaCI2, suggesting the presence of an acyl hydrolase and phospholipase D activities. Differential centrifugation studies indicate that 50 to 80% of the crude homogenate's phospholipase D activity is membrane-bound. Density centrifugation experiments suggest that the membranebound phospholipase D activity is localized primarily on mitochondrial membranes.Our interest in phospholipid degradation originates from a desire to minimize the loss of lipid constituents from membranes during isolation from plant tissues. Inactivation of membrane-bound CCR' from wheat aleurone and the vanadate-sensitive proton pump from maize roots during isolation was associated with degradation of membrane lipids (2, 28). lipids by a reconstitution restored transport activity to initial levels (2). Such results indicated that net proton transport was inhibited because of increased leakage of proton resdulting from phospholipid degradation. Losses in membrane phospholipids from corn root microsomes did not correspond to an accumulation of either lysophospholipids or PA (2), suggesting that neither PLase A2 nor D was responsible for the degradation. However, the exact nature of the lipolytic enzymes responsible for the phospholipid degradation in maize root homogenates has not been identified and is the focus of this report. Results from this study indicate the presence of at least two lipolytic enzyme activities, a Ca-stimulated PLase D and an acyl hydrolase. MATERIALS AND METHODS Preparation of Crude Homogenates and Membrane FractionsRoots (40-50 g fresh weight) from 3 d old maize seedlings (Zea mays L. cv WF9 x Mol7) were grown on filter paper moistened with 0.1 mm CaCl2 and harvested as described previously (19). Roots were homogenized by mortar and pestle for 3 min in the presence of 50 mm Mes titrated to pH 6.0 with BTP, 0.25 M sucrose, and 5 mM DTT using 3 mL of buffer per g of roots at 0 to 4°C. The brei was filtered through four layers of cheesecloth, and the resulting filtrate was used as the crude homogenate. Homogenates and membranes prepared as above contained less than 1 ,AM Ca2' as determined by Ca-selective electrode (data not shown). Protein concentration was determined after precipitation by TCA in the presence of deoxycholate by a modification of the Lowry method (1).When membranes in the pH 6.0 crude homogenate wer...

The influence of poly(L‐lysine) binding on the coupled activities of nitrate‐sensitive H+‐ATPase in isolated corn (Zea mays L. cv. FRB73) root tonoplast vesicles was investigated. The addition of membrane‐impermeable poly(L‐lysine) caused a slow increase in light scattering of the tonoplast suspension. Electron microscopy showed that the increase was the result of an aggregation of the vesicles. In the presence of 75 mM KCl, a concentration sufficient to sustain near optimal ATP hydrolysis, poly(L‐lysine) slightly enhanced the hydrolysis activity but significantly inhibited proton pumping of the H+‐ATPase. Inhibition increased with the average molecular mass of poly(L‐lysine) and reached a maximum at 58 kDa. When total osmolarity was kept constant, the replacement of sucrose by KCl enhanced both ATP hydrolysis and proton pumping activities. However, enhancement of proton pumping was significantly greater than that of ATP hydrolysis. An increase in KCl, but not K2SO4, significantly relieved poly(L‐lysine)‐induced inhibition of proton pumping. Kinetic analysis indicated that poly(L‐lysine) did not significantly affect the proton leakage of the tonoplast membranes under different energetic conditions. These results suggest that the electrostatic interaction between poly(L‐lysine) and the negative charges on the exterior surface of tonoplast vesicles could change the coupling ratio of ATP hydrolysis to proton pumping. Thus, the surface charge of the tonoplast membrane may be involved in the regulation of these two activities.