John L. Giannini

Pyridine nucleotide oxidation by a plasma membrane fraction from red beet (Beta vulgaris L.) storage tissue

The potential role of pyridine nucleotide oxidation in the energization and/or regulation of membrane transport was examined using sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue. In this system, pyridine nucleotide oxidation, which was enhanced in the presence of ferricyanide, occurred. In the presence or absence of ferricyanide, the oxidation of NADH was several-fold greater than the oxidation of NADPH, indicating that it was the preferred substrate for oxidation in this system. Ferricyanide reduction coupled to NADH oxidation did not require the transmembrane movement of reducing equivalents since ferricyanide incorporated inside the vesicles could not be reduced by NADH added externally to the vesicles, unless the vesicles were made leaky by the addition of 0.05% (v/v) Triton X-100. Using fluorescent probes for the measurement of transmembrane pH gradients and membrane potentials, it was determined that NADH oxidation did not result in the production of a proton electrochemical gradient or have any effect upon the proton electrochemical gradient produced by the plasma membrane H+-ATPase. The oxidation of NADH in the presence of ferricyanide did result in the acidification of the reaction medium. This acidification was unaffected by the addition of Gramicidin D and stimulated by the addition of 0.05% (v/v) Triton X-100, suggesting a scalar (nonvectorial) production of protons in the oxidation/reduction reaction. The results of this study suggest that the oxidation of pyridine nucleotides by plasma membrane vesicles is not related to energization of transport at the plasma membrane or modulation of the activity of the plasma membrane H+-ATPase.

A small scale procedure for the isolation of transport competent vesicles from plant tissues.

A microscale method for the isolation of selectively sealed microsomal membrane fractions from plant tissue is presented. The method is based on differential centrifugation in a table top microcentrifuge to accommodate small sample size (10-25 g tissue) and the addition of KI or KCl in the homogenization medium for isolating selectively sealed plasma membrane or tonoplast vesicles. This microscale procedure was found to be useful in isolating membranes from red beet (Beta vulgaris) storage tissue, sugar beet (Beta vulgaris) storage tissue, corn (Zea mays) roots, and soybean (Glycine max) roots. This paper also describes the ability to further purify an enriched red beet plasma membrane fraction on a discontinuous sucrose density gradient, in a microcentrifuge, that is highly competent in ATP-dependent H+-transport. The speed and wide applicability of this procedure make it ideal when a large number of samples need to be processed.

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea: II. Partial characterization of Ca2+ transport and glyceollin effects

Calcium uptake was examined in sealed plasma membrane vesicles isolated from the plant pathogenic fungus, Phytophthora megasperma f. sp. glycinea. Calcium uptake was ATP-dependent and by the addition of various ionophores in the presence of ATP, it was shown that Ca2+ transport was mediated by a nH+/Ca2+ antiport. Further evidence for this antiport mechanism included Ca2+ uptake driven by an imposed pH gradient and the observation that calcium could dissipate a steady-state pH gradient across the vesicle membrane. Transport mediated by the nH+/Ca2+ antiport was optimal at pH 7.0, and demonstrated saturation kinetics for Ca2+ with a Km of about 7 microM. Glyceollin, a soybean phytoalexin, was found to inhibit Ca2+ transport consistent with its ability to increase H+ conductance. In the presence of glyceollin, calcium leakage from Phytophthora membrane vesicles also increased. This study provides basic information about calcium transport in a plant pathogenic fungus as well as demonstrating a possible mode of action of a phytoalexin.

The effect of glyceollin on proton leakage in Phytophthora megasperma f. sp. glycinea plasma membrane and red beet tonoplast vesicles

Abstract Proton transport in sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea was inhibited by glyceollin. At 50 μM glyceollin no proton transport could be observed. Glyceollin at this concentration did not significantly affect the H + -ATPase responsible for proton pumping at the plasma membrane. Passive proton conductance increased significantly in the presence of glyceollin. These results suggest that glyceollin may inhibit fungal growth by diminishing the proton gradient necessary for solute uptake. Inhibition of the proton gradient appears to be due to increased proton conductance across the membrane in the presence of glyceollin and not inhibition of the H + -ATPase. Glyceollin also caused increased passive proton conductance in red beet ( Beta vulgaris L.) tonoplast vesicles at levels where the ATPase was minimally inhibited. These data taken together suggest that a primary phytotoxic effect of glyceollin may be to cause proton or ion leakage across membranes.

Fluoride effects on the plasma membrane ATPase of sugarbeet

Abstract Fluoride, a common air pollutant long known as a toxicant to many plant processes, inhibits mitochondrial, chloroplast and tonoplast ATPases. In the present study, the effects of fluoride at various substrate concentrations on the plasma membrane ATPase of sugarbeets ( Beta vulgaris L.) were investigated. The plasma membrane ATPase was inhibited by lower concentrations (5 mM) of fluoride than the above indicated ATPases. The amount of inhibition due to fluoride increased with increasing concentrations of free Mg 2+ in the reaction medium. The data suggest that fluoride inhibition of the plasma membrane ATPase is at the active site of the enzyme and occurs via a magnesium-fluoro-complex.

Effects of phaseollin on membrane leakage in red beet vacuoles and tonoplast vesicles

Abstract Acting in a manner similar to that of glyceollins I and III, phaseollin appears to inhibit proton transport in red beet tonoplast vesicles. In the presence of 80 μM phaseollin, proton transport in tonoplast vesicles was inhibited by >90%. This inhibition appears to be due to increased conductance across the tonoplast membrane and not to inhibition of tonoplast ATPase. Moreover, phaseollin was shown to cause molecules, e.g. β-anthocyanidin, to leak across the vacuolar membrane.

The effect of glyceollin on soybean (Glycine max L.) tonoplast and plasma membrane vesicles

Abstract An improved method for the isolation of transport-competent plasma membrane and tonoplast vesicles from soybean (Glycine max L., Co. Harsoy) is presented. When resolved on linear sucrose gradients, the soybean membrane vesicles displayed two peaks of ATP-dependent H+-transport activity that were inhibited by either vanadate (plasma membrane) or nitrate (tonoplast). That the vanadate and nitrate sensitive H+-transport activities were representative of plasma membrane and tonoplast vesicles was further supported by their differing pH optima for ATP-dependent H+-transport. Membrane vesicles displaying vanadate sensitive H+ transport showed optimal activity at pH 6.5 while membrane vesicles displaying nitrate sensitive H+-transport showed optimal activity at pH 7.5. When ATP-dependent H+-transport was examined as a function of Mg:ATP concentration, both vesicle types displayed simple Michaelis-Menten type kinetics and Km-values of 0.56 and 0.97 mM were observed for plasma membrane and tonoplast vesicles, respectively. Incubation of plasma membrane or tonoplast vesicles with glyceollin resulted in an inhibition of ATP-dependent H+-transport. While ninety percent inhibition of ATP-dependent H+-transport was observed with tonoplast vesicles when incubated with 25 μM glyceollin, a 4-fold higher concentration of the phytoalexin was required to produce a similar inhibition with plasma membrane vesicles. Although glyceollin had only small effects upon ATP hydrolytic activity, this compound increased the H+-conductance of the vesicles in a manner consistent with the relative inhibition of ATP-dependent H+-transport.

The effect of rishitin on potato tonoplast vesicle and vacuole proton transport

Rishitin, a known potato phytoalexin, was tested for its effects on proton transport. Like the pterocarpan phytoalexins, glyceollin and phaseollin, rishitin was found to inhibit proton transport. At 100 microM rishitin, proton transport in potato tonoplast vesicles was inhibited by > 95%. This inhibition appears to be due to an increase in proton conductance and not to inhibition of the tonoplast ATPase. Potato vacuoles were also shown to have increased proton leakage in the presence of rishitin.

The effect of assay composition, detergent solubilization and reconstitution on red beet (Beta vulgaris L.) plasma membrane H+-ATPase kinetic properties

Modification of the salt concentration, composition and/or buffer type in the assay of plasma membrane ATPase activity caused substantial changes in the Km and slight changes in the temperature dependence of this enzyme. The Km and temperature dependence were also affected by detergent solubilization of the ATPase and its subsequent reconstitution into liposomes. Modulation of kinetic properties by assay composition and hydrophobic state reflect the sensitivity of the plasma membrane H+-ATPase to its immediate environment. This may indicate a possible regulatory mechanism for this important plant enzyme.