The responses of the vacuolar membrane (tonoplast) protonpumping inorganic pyrophosphatase (HI-PPase) from oat (Avena sativa L.) roots to changes in Mg2" and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the be conserved as a proton gradient if the H+-PPase functions in vivo as a pump (14,22,29). Insight into the conditions under which the H+-PPase operates in vivo, and therefore into its function, would be gained if the substrate and other modulators of its activity were known.In vitro, vacuolar H+-PPases require both Mg2' and K+, in addition to PPi, for complete activity (7,20,26,27,29). However, the response of the enzyme to changes in both [PPi]0to and [Mg],,, can be complex (11,14,27,29). In a reaction medium containing PPi, Mg2", and K+, the complexes and ions present include free Mg, free PPi, MgPPi, Mg2PPi, K+, and KPPi as well as various protonated forms of the complexes (e.g. 11, 29). This makes it very difficult to test the effect of individual complexes on the activity of the H+-PPase because the concentration of any single complex cannot easily be changed without altering the concentration of some others. Nonetheless, previous kinetic studies have suggested that the enzyme is activated by both Mg2" and K+ ions and that the substrate is MgPPi (11,26,29). In addition, both free PPi and Mg2PPi might inhibit the enzyme, although this appears to depend on the tissue from which the tonoplast membranes are prepared (11,27,29).One limitation of the work reported thus far is that the validity of the conclusions drawn from kinetic experiments in vitro has not been tested by quantitative models of the observed data. This paper reports detailed descriptions of the response of the oat (Avena sativa L.) root vacuolar H+-PPase to changes in [Mg]

The HW-translocating inorganic pyrophosphatase (H -PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca2" and a number of other divalent cations (Co2, Mn2", Zn2+ ATPase2 (EC 3.6.1.3) and a H+-PPase (EC 3.6.1.1) (28). The two pumps have been purified to near-homogeneity by independent techniques (6,18,25,29).It is now clear that both the vacuolar H+-ATPase and H+-PPase mediate electrogenic H+ translocation into the same intracellular compartment. Individual mechanically isolated vacuoles display both ATP-and PPi-dependent inward currents (12), and the steady-state transmembrane pH difference and electrical potential difference generated by the ATPase and PPase in isolated vacuolar vesicles interact nonadditively and are subject to kinetic control by a common H+-electrochemical potential difference (14,28). The continuous and simultaneous operation of both pumps in vivo might, however, appear redundant. Therefore, it is important to know whether the two enzymes are under independent cytoplasmic control. This question is relevant not only for tonoplast energization, but also for gaining a deeper understanding of the general metabolic role of PPi.PPi is becoming increasingly recognized as an important metabolite in plant cells, where it is present in the cytosol at concentrations of between 200 and 300 Mm (32, 34). Consideration of two of the best-characterized (soluble) PPi-dependent enzymes in plants-UDP-glucose pyrophosphatase and PPi fructose-6-P-1-phosphotransferase-indicates that modulation of cellular PPi levels could have profound metabolic consequences. Thus, the cytosolic reactions catalyzed by UDP-glucose pyrophosphatase and PPi fructose-6-P-1-phosphotransferase are close to equilibrium in physiological conditions (34). Diminished cytosolic PPi would therefore be expected to favor the formation of fructose-6-P from fructoseThe vacuolar membrane (tonoplast) of higher plant cells contains two primary H+ pumps: a vacuolar-type H+-

Summary. H*-coupled transport in plant and fungal cells is relatively insensitive to external pH (pHo). H+-coupled C1-transport at the plasma membrane of Chara corallina was studied to explore the phenomena responsible for this insensitivity. Raising pH, from a control value of 7.5 to 9.0 results in a modest (2.5-fold) decline in Jmax and increase in K,,. Further increase in pHo results in a selective increase in Jm~x, in accordance with predictions from a reaction kinetic model of the transport system (Sanders, D., Hansen, U. The results are discussed in terms of the general physiological requirement that fluxes through H ~-coupled transport systems are relatively insensitive to environmental variation in pHo. It is proposed that (i) the weak (but finite) dependence of pHc on pHo, coupled with (ii) the strong dependence of H+-coupled transport on pHc are instrumental in endowing H+-coupled transport systems with a relative insensitivity to variation in pHo. This hypothesis might also explain why pHc in plants and fungi is not acutely controlled with respect to variation of pHo.

Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An Proton transport plays a central role in energy transduction in plants (11). Primary H+ transport at the energy-coupling membranes of chloroplasts and mitochondria is driven by light and redox potential energy, respectively, and results in the formation of a transmembrane electrochemical H+ gradient (AdZH+).3 A dissipative flow of protons down this gradient is then coupled to synthesis of ATP. At the plasma membrane and tonoplast, hydrolysis of phosphoanhydride bonds is used to energize H + transport, with reverse flow of H+ down the resulting (A-AH+. wering transport of other solutes through discrete secondary systems.Classical electrophysiological techniques have given insight into the kinetics of H + transport across the plasma membrane of intact plant cells (1) and patch clamp executed in a 'whole cell mode' enables the study of ATP-dependent H + currents in intact vacuoles (4, 12). More commonly, however, H+ transport is studied in isolated membrane vesicles, whose small size prohibits the electrophysiological approach. Further, the fact that 3H rap- ' With the exception of nonsteady state investigations of photosynthetic systems in the ms time range (14), the kinetics of vesicular transmembrane H + translocation have been calculated from analog recordings. Thus, rates have been estimated from hand-drawn tangents to curves-a process which is both laborious and errorsome. The problem is particularly acute for secondary transport where the pH gradient is often generated artificially: because of the intrinsic permeability of the membrane to H+ and the high surface area:volume ratio of vesicles, secondary transport is often initiated against a shifting baseline and the signal obtained is short-lived owing to depletion of the limited reserves of intravesicular protons.Here we report a method for the logging and subsequent analysis of fluorescence data from ApH-reporting probes in membrane vesicles. Using tonoplast vesicles from Beta vulgaris L. as a model, we show that time-dependent fluorescence change resulting from activation of primary and secondary H+ transport systems can be simply and accurately estimated by least squares fitting of single exponential functions to digitized, stored data. A preliminary report of this work has appeared previously (13). MATERIALS AND METHODS

The effects of Cl- concentration and membrane potential (delta psi) on Cl- influx in isolated vesicles of vacuolar membrane (tonoplast) from red beet (Beta vulgaris L.) storage tissue have been characterized using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). The initial rate of Cl- transport into the vesicles was enhanced both by the imposition of a positive delta psi and by increases in extravesicular Cl- concentration. The kinetic mechanism underlying these responses was investigated by examining the accuracy with which the data could be described by several transport models. A model based on constant field theory yielded a poor description of the data, but satisfactory fits were generated by pseudo-two-state reaction kinetic models based on classical carrier schemes. Fits were equally good when it was assumed that charge translocation accompanied Cl- entry, or when charge was carried by the unloaded transport system, as long as only a single charge is translocated in each carrier cycle. Expansion of the models to three states enabled description of the Cl- concentration dependence of transport by changes in a single, voltage insensitive rate constant which is tentatively identified with Cl- binding at the external surface of the membrane. The derived value of the dissociation constant between Cl- and the transport system is estimated at between 30 and 52 mM.

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