Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

How do protons cross the membrane-solution interface? Kinetic studies on bilayer membranes exposed to the protonophore S-13 (5-chloro-3-tert-butyl-2′-chloro-4′ nitrosalicylanilide)

Summary A simple carrier model describes adequately the transport of protons across lipid bilayer membranes by the weak acid S-13. We determined the adsorption coefficients of the anionic, A^–, and neutral, HA, forms of the weak acid and the rate constants for the movement of A^– and HA across the membrane by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. These measurements agree with the results of voltage clamp and charge pulse kinetic experiments. We considered three mechanisms by which protons can cross the membranesolution interface. An anion adsorbed to the interface can be protonated by (i) a H⁺ ion in the aqueous phase (protolysis), (ii) a buffer molecule in the aqueous phase or (iii) water molecules (hydrolysis). We demonstrated that the first reaction cannot provide the required flux of protons: the rate at which H⁺ must combine with the adsorbed anions is greater than the rate at which diffusion-limited reactions occur in the bulk aqueous phase. We also ruled out the possibility that the buffer is the main source of protons: the rate at which buffers must combine with the adsorbed anions is greater than the diffusion-limited rate when we reduced the concentration of polyanionic buffer adjacent to the membrane-solution interface by using membranes with a negative surface charge. A simple analysis demonstrates that a hydrolysis reaction can account for the kinetic data. Experiments at acid pH demonstrate that the transfer of H⁺ from the membrane to the aqueous phase is limited by the rate at which OH combines with adsorbed HA and that the diffusion coefficient of OH^– in the water adjacent to the bilayer has a value characteristic of bulk water. Our experimental results demonstrate that protons are capable of moving rapidly across the membrane-solution interface, which argues against some mechanisms of local chemiosmosis.     

The kinetic mechanism by which CCCP (carbonyl cyanidem-Chlorophenylhydrazone) transports protons across membranes

Summary We demonstrate that a simple kinetic model describes the transport of protons across lipid bilayer membranes by the weak acid CCCP (carbonyl cyanidem-chlorophenylhydrazone). Four parameters characterize this model: the adsorption coefficients of the anionic and neutral forms of the weak acid onto the interface ( _A and _HA) and the rate constants for the movement of A^– and HA across the membrane (k _A andk _HA). These parameters were determined by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. From these equilibrium and steady state measurements on diphytanoyl phosphatidylcholine/chlorodecane membranes we found that _A= _HA=1.4 10^-3cm,k _A=175 s^–1 andk _HA=12,000 sec^–1. These parameters and our model describe our kinetic experiments if we assume that the protonation reactions, which occur at the interfaces, remain at equilibrium. The model predicts a single exponential decay of the current in a voltage-clamp experimetn. The model also predicts that the decay in the voltage across the membrane following an intense current pulse of short duration (50 nsec) can be described by the sum of two exponentials. The magnitudes and time constants of the relaxations that we observed in both voltage-clamp and charge-pulse experiments agree well with the predictions of the model for all values of pH, voltage and [CCCP].     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).     

Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H⁺ transport was evaluated by measuring H⁺-dependent accumulation of acridine orange (AO). Active H⁺ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K _m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H⁺ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H⁺ accumulatioin was abolished by protonophores. such as nigericin (10^–6 mol/liter) or CCCP (10^–5 mol/liter), and by inhibitors of nonmitochondria H⁺ ATPase, such as NEM or NBD-Cl, at a concentration of 10^–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H⁺ pumps, such as DCCD (10^–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H⁺ transport by about 90%. Vanadate (2×10^–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H⁺ transport. The stilbene derivative DIDS (10^–4 mol/liter), which inhibits anion transport systems, abolished H⁺ transport completely. MgATP-dependent H⁺ transport was found to be anion dependernt in the sequence Cl^–>Br^–>gluconate^–; in the presence of SO ₄ ^–2 . CH₃COO^– or No ₃ ^– , no H⁺ transport was observed. MgATP-dependent H⁺ accumulation was also cation dependent in the sequence K⁺>Li⁺>Na⁺=choline⁺, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl^– and a K⁺ conductance, as well as a small H⁺ conductance. were found in the microsomal membranes. When membranes containing the H⁺ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H⁺ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H⁺ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.     

Is the ATP — dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump

Summary Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37°C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K⁺), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH₄Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37°C, but not at 0°C. The Mg^–+-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl₂ in the presence of 1 mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H^– ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.Abbreviations Mops 2-(N-morpholine)-propanesulfonic acid - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-Dinitrophenol - EDTA Ethylenediaminetetracetic acid