Ronald J. Poole

Vacuolar H+-translocating pyrophosphatases: a new category of ion translocase

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.

The computed free energy change of hydrolysis of inorganic pyrophosphate and ATP: apparent significance. for inorganic-pyrophosphate-driven reactions of intermediary metabolism

Abstract Energy from cytosolic hydrolysis of inorganic pyrophosphate (PP i ) can be conserved through PP i acting as a specific phosphoryl donor or through the substitution of reactions involving PP i for those involving ATP. To assess the relative importance of PP i in this context, the standard free energy change ( ΔG °′) and hence the actual free energy change ( ΔG ) values for ATP and PP i hydrolysis have been computed for a simple model cytosol. ΔG °′ ATP and ΔG °′ PP i show dissimilar responses to pH and free Mg 2+ , with implications for cell energetics during anaerobiosis. The impact of the estimates on possible in vivo function of two primary H + -translocating phosphoanhydrolases (an ATPase and PPase) at the vacuolar membrane of higher plants is discussed.

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts

SummaryThe voltage- and time-dependent K+ current,IK+out, elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La3+) to the extracellular medium. These results suggested that the influx of external Ca2+ was necessary for K+ current activation. The IC50, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 μm at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca2+ actuated the K+ current, we altered either the Ca2+ buffering capacity or the free Ca2+ concentration of the intracellular medium (pipette filling solution). By these means,IK+out could be varied over a 10-fold range. Increasing the free Ca2+ concentration from 40 to 400nm also shifted the activation of the K+ current toward more negative potentials. Maintaining cytoplasmic Ca2+ at 500nm with 40nm EGTA resulted in a more rapid activation of the K+ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca2+ on depolarization. Increasing intracellular Ca2+ to 500nm or 1 μm also led to inactivation of the K+ current within a few minutes. It is concluded thatIK+out is regulated by cytosolic Ca2+, which is in turn controlled by Ca2+ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.

Electrogenic pump activity in red beet: Its relation to ATP levels and to cation influx

SummaryIn storage tissue ofBeta vulgaris L., carbonyl cyanidem-chlorophenylhydrazone or cyanide+salicylhydroxamic acid reduce cell electropotentials from about −200 to below −100 mV. The relationship between potential and cellular ATP level is examined during treatment with different concentrations of inhibitiors. At low ATP levels the potential rises sharply with increases in ATP, but above an ATP level of approximately 50% of the uninhibited level the potential changes very little with ATP concentration. A plot of membrane potentialvs.86Pb+ influx or of potentialvs. net K+ uptake indicates that as the level of inhibition is decreased, the potential tends to reach a limit while cation influx and net uptake continue to increase. Resistance measurements, although subject to difficulties of interpretation, indicate no change in conductance with potential, ion flux, or ATP level. Thus the membrane potential should directly reflect electrogenic pump activity, attributed to active uncoupled H+ efflux. K+ uptake can occur against its electrochemical gradient and is attributed to a coupled K+ influx/H+ efflux pump. The results show that the electrogenic pump activity is independent of the K+/H+ exchange rate. Thus electrogenic H+ efflux and K+/H+ exchange may represent different transport systems, or different modes of operation of a single pump with variable stoichiometry.

[12] Preparation of tonoplast vesicles: Applications to H+-coupled secondary transport in plant vacuoles

Publisher Summary This chapter describes a method for the preparation of tonoplast vesicles. The membranes collected from the 10%–23% sucrose have two predominant enzyme activities, an ATPase and a PPase, which are qualitatively similar to those of isolated intact vacuoles. Marker enzyme activities indicate only small amounts of other identifiable membranes in the tonoplast vesicle fraction. Various applications to H + -coupled transport are discussed. For a substrate to be transported across a membrane, both a pathway and a driving force are required. Two transport systems serve to build up the proton motive force (PMF) by the direct utilization of metabolic energy (ATP or PP i ). Active transport of cations through antiport mechanisms across the tonoplast operates by coupling the flux of cations to the opposite flux of H + . The measurement of H + -coupled transport requires, then, prior loading of the vesicles with one of the exchangeable substrates (cation or H + ).

Pharmacology of the Ca2+-dependent K+ channel in corn protoplasts

We investigated the sensitivity of the Ca2+‐dependent K+ current, I K (Ca), present in corn protoplasts, to different K+ channel blockers. I K(Ca) was inhibited by external Cs+ (10 mM), Ba2+ (10 mM), and quinine (0.5 mM): reagents which block many types of outward‐rectifying K+ channels. In contrast 4‐aminopyridine (5 mM), an inhibitor of delayed rectifier or inactivating K+ currents, had no effect. Neither of the peptide toxins, apamin or charybdotoxin, specific for Ca2+‐dependent K+ channels in animal cells, inhibited currents when used in the nanomolar concentration range. However, higher levels of charybdotoxin (10 μM) caused marked reduction of I K(Ca).

Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.

Vacuolar H+-translocating Inorganic Pyrophosphatase: Biochemistry and Molecular Biology

General acceptance of the chemiosmotic hypothesis (Mitchell 1961) has given rise to the view that membrane-bound H+-pumps constitute the primary transducers by means of which living cells inter-convert light, chemical and electrical energy. Through the establishment and maintenance of trans-membrane electrochemical gradients, H+ pumps energise the transport of other solutes or, in the special case of the energy-coupling membranes of mitochondria, chloroplasts and bacteria, transduce the H+-electrochemical gradient generated by membrane-linked anisotropic redox reactions to the synthesis of ATP (Harold, 1986). Given the multitude of biological reactions energised by ATP, primary H+-translocation and the inter-conversions of ATP have come to be recognised as the principal generators of usable energy in the cell. Against this background it is, therefore, surprising to find that the vacuolar membrane (tonoplast) of plant cells contains not only a V-(“vacuolar”) type H+ -ATPase (EC 3.6.1.3) (Rea and Sanders, 1987; Nelson and Taiz, 1989) but also an inorganic pyrophosphate- (PPi) energised H+ pump (V-Type H+-PPase; EC 3.6.1.1) (Rea and Sanders, 1987). Both enzymes catalyse inward electrogenic H+-PPase has the unusual characteristic of exclusively utilising PPi as energy source (Rea and Poole, 1986).

Transport of Inorganic Ions in Tonoplast Vesicles

Since the tonoplast proton pumps create a positive membrane potential in the vacuole, the maintainance of high cytoplasmic K+ and the accumulation of Cl− and NO3 − in the vacuole requires only passive transport mechanisms (channels or uniports) for these ions, energized by ∆Ψ. On the other hand, the vacuolar accumulation of Na+ and Ca2+, and the retrieval of stored nutrient anions such as NO3 − for metabolic use requires active transport mechanisms, which we now believe to be energized by proton efflux from the vacuole. This paper will discuss our work on these proton-coupled secondary transport mechanisms, with emphasis on their physiological significance, the technical problems involved, and the many questions remaining to be investigated.

Proton Pump and ATPase Activities in Tonoplast Vesicles from Storage Tissue of Red Beet

Preparations of sealed membrane vesicles represent an ideal experimental system for studying mechanisms of membrane transport (Turner, 1983 for review). With the development of the methodology to isolate sealed membrane vesicles from plant cells by Sze (1980), this valuable tool could then be applied to the study of transport across plant membranes. Using this system, a number of laboratories have recently characterized ATP-dependent H+ transport in membrane vesicles isolated from homogenates of plant tissue (Hager et al. 1980; Rasi-Caldogno et al. 1981; Dupont et al. 1982; Mettler et al. 1982; Bennett and Spanswick 1983; Churchill et al. 1983). From these studies, it appears that two distinct types of H+-transporting ATPase can be observed in plant vesicle preparations (Churchill et al. 1983 and references therein). One type of ATPase is stimulated by anions, inhibited by N03 - and insensitive to vanadate while the other type of ATPase is stimulated by cations and inhibited by vanadate (Churchill et al. 1983 and references therein). Both types of H+-transporting ATPase are insensitive to oligomycin and NaN3 (Sze, 1983; Dupont et al. 1982; Churchill and Sze, 1983) which indicates that they are not representative of mitochondrial ATPase. These two types of H+-transporting ATPase can be separated on sucrose or dextran density gradients (Churchill et al. 1983) which suggests that they are associated with different membrane components of the plant cell.