Kinetic Analysis of Proton Transport by the Vanadate-Sensitive ATPase from Maize Root Microsomes

Abstract

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...

Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes

“…The passive movement of ions was based on the Fick's diffusion law combined with the Goldman-Hodgkin-Katz current equation for electrodiffusive flux. Similarly, this approach predicted with high degree of accuracy the magnitudes of the ionic fluxes in vesicles in which the proton gradients were generated by ATP-driven proton pumps [21][22][23][24]. Finally, the membrane potential was obtained considering the bilayer as a capacitor and its value was obtained from the variation of the net current across the membrane with time.…”

“…In these systems, fluorescent dyes sensitive to pH were used to follow the movement of protons and the kinetic models were determined by correlating the pH variations, ion fluxes and electric potential across the membrane of the liposomes [21][22][23][24].…”

Effects of Solubilization on the Inhibition of the P-Type ATPase from Maize Roots by N-(Ethoxycarbonyl)-2-Ethoxy-1,2-Dihydroquinoline

“…The rate of ATP hydrolysis by the canine kidney (Na + K) ATPase (Sigma Chemical Co.) was also determined by the release of inorganic phosphate as described previously (10). The assay media contained 20 mi Hepes (pH 7.5), 100 mm NaCl, 25 mM KCl, 5 Where indicated, the EEDQ treatment was terminated by diluting the vesicles 5-to 10-fold with ice-cold 20 mm Hepes resuspension buffer.…”

“…Bars indicating the magnitude of the SE are included in figures when the variation exceeds the size of the data symbol. Proton transport was followed by changes in the absorbance of 7.5 jLM acridine orange as described previously (10) in an assay buffer identical to that described above for ATP hydrolysis. The initial rate of proton transport was calculated as described previously (10).…”

“…Microsomes were isolated from 3-d-old roots by differential centrifugation as described previously (8), except that the 25 mm bis-trispropane (pH 7.8) in the homogenizing buffer was replaced with 50 mm Hepes (pH 7.5). Microsomes were suspended in 5 mL of homogenizing buffer and layered over a gradient consisting of two 10-mL steps of 34 and 42% (w/w) sucrose in 5 mm Hepes (pH 7.5).…”