Plant Physiol. (1990) 93, 1063-1070 Received for publication December 18, 1989 0032-0889/90/93/1 063/08/$01 .00/0 Accepted March 15, 1990 Substrate Kinetics of the Tonoplast H+-Translocating Inorganic Pyrophosphatase and Its Activation by Free Mg2+ 1
Philip J. White2, Jacqueline Marshall3, and J. Andrew C. Smith` Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, United Kingdom
ABSTRACT Solute accumulation in plant vacuoles appears to be ener- gized by two distinct H+-translocating enzymes at the tono- To clarify the kinetic characteristics and ionic requirements of plast, an H+-ATPase and an H+-PPiases (11, 25). The tono- the tonoplast H+-translocating inorganic pyrophosphatase (H+- PPiase), PPi hydrolysis and PPi-dependent H+ transport were plast H+-ATPase has been studied extensively, and its kinetic studied in tonoplast vesicles isolated from leaf mesophyll tissue characteristics are relatively well established (1, 25, 30). In of Kalanchoe daigremontiana Hamet et Perrier de la Bathie. The contrast, the kinetics of the H+-PPiase are not yet completely tonoplast H+-PPiase showed an absolute requirement for a mon- understood, mainly because of the difficulty of varying inde- ovalent cation and exhibited hyperbolic kinetics with respect to pendently the concentrations of potential substrates and ef- cation concentration. H+-PPiase activity was maximal in the pres- fectors when assaying this enzyme. However, a more complete ence of K+ (K50 approximately 3 millimolar), with PPi-dependent kinetic description of the tonoplast H+-PPiase would be use- H+ transport being more selective for K+ than PPi hydrolysis. ful in elucidating the physiological role of this enzyme and When assayed in the presence of 50 millimolar KCI at fixed PPi in explaining how its activity might be modulated in vivo concentrations, H+-PPiase activity showed sigmoidal kinetics with (17, 25, 31). respect to total Mg concentration, reflecting a requirement for a Of the properties described thus far for the tonoplast H+- Mg/PPi complex as substrate and free Mg2+ for activation. At PPiase, there is general agreement that the enzyme is stimu- saturating concentrations of free Mg2+, H+-PPiase activity exhib- lated greatly by K+ and, to a lesser extent, by other alkali- ited Michaelis-Menten kinetics towards MgPPi2- but not Mg2PPi, metal cations (19, 24, 34, 35). H+-PPiase activity is also Mg- demonstrating that MgPPi2- was the true substrate of the enzyme. dependent, and when assayed at fixed PPi concentrations The apparent Km (MgPPi2-) for PPi hydrolysis (17 was micromolar) exhibits a to significantly higher than that for PPi-dependent H+ transport (7 usually sigmoidal response increasing total Mg micromolar). Free Mg2+ was shown to be an allosteric activator concentrations (5, 32, 34, 35). On the other hand, in the of the H+-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis presence of saturating Mg, the activity of the H+-PPiase and 2.7 for PPi-dependent H+ transport. Half-maximal H+-PPiase typically shows hyperbolic kinetics with respect to total PPi activity occurred at a free Mg2+ concentration of 1.1 millimolar, concentration, with an apparent Km for total PPi between 5 which lies within the range of accepted values for cytosolic Mg2+. and 30 ,M (5, 10, 13, 19, 20, 23 24, 35). These observations In contrast, cytosolic concentrations of K+ and MgPPi2- appear to suggest that an Mg/PPi complex is the true substrate for the be saturating for H+-PPiase activity. We propose that one function enzyme, and both MgPPi2- (13, 34, 35) and Mg2PPi (34) have of the H+-PPiase may be to act as an ancillary enzyme that been proposed as possible substrates. In fact, the substrate maintains the proton-motive force across the vacuolar membrane kinetics of the tonoplast H+-PPiase have been difficult to when the activity of the tonoplast H+-ATPase is restricted by interpret unambiguously, and there appear to be species dif- substrate availability. As ATP levels decline in the cytosol, free ferences in the effects ofsupraoptimal Mg/PPi concentrations, Mg2+ would be released from the MgATP2- complex, thereby which markedly reduce PPiase activity in some preparations activating the tonoplast H+-PPiase. (4, 10, 13, 17, 32, 35), but not in others (5, 19, 34). By analogy with the cytoplasmic PPiases ofmicroorganisms (7, 14), it has also been suggested that the activity of the tonoplast H+-PPiase is strongly modulated by certain effec- ' Supported by a grant from the Science and Engineering Research tors. In particular, free Mg2' has been proposed as an activator Council, UK. of the enzyme (13, 34, 35) and free PPi (i.e. PPi4-, HPPi3-, 2 Present address: Botany School, University ofCambridge, Down- or H2PPi2-) as a competitive inhibitor (34, 35), with Mg2PPi ing Street, Cambridge, CB2 3EA, UK. also possibly being inhibitory at high concentrations (13). 3Present address: AFRC Institute of Arable Crops Research, However, a complete kinetic description of the tonoplast H+- Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, UK. sAbbreviations: H+-PPiase, H+-translocating inorganic pyrophos- 4 Present address and address for correspondence: Department of phatase; BTP, 1,3-bis-[tris(hydroxymethyl)methyl-amino]propane; Plant Sciences, University of Oxford, South Parks Road, Oxford, quinacrine, 6-chloro-9-{[4-(diethylamino)-1-methylbutyl]amino -2- OX I 3RB, UK. methoxyacridine dihydrochloride. 1063 1 064 WHITE ET AL. Plant Physiol. Vol. 93, 1990
PPiase that quantitatively accounts for the influence of Mg2", PPi hydrolysis were assayed over 20 min. Rates of PPi hy- free PPi, MgPPi2-, and Mg2PPi on enzyme activity has not drolysis were linear during the assay and were corrected for yet been presented. Thus, in the present work we have studied background rates of nonenzymic substrate hydrolysis deter- the tonoplast H+-PPiase of leaf mesophyll cells of the CAM mined using boiled preparations. plant Kalanchoe daigremontiana, a species showing high H+- Initial rates of PPi-dependent H+ transport into tonoplast PPiase activity (19, 37), using assay conditions that allowed vesicles were assayed at 25°C by following the fluorescence us to vary independently the concentrations of potential quenching of quinacrine as previously described (37). Quin- substrates and effectors. Our results suggest that free PPi and acrine fluorescence was monitored using a Perkin-Elmer LS- Mg2PPi have little effect on the enzyme, and that the activity SB luminescence spectrometer (Perkin-Elmer Ltd., Beacons- ofthe tonoplast H+-PPiase of K. daigremontiana can be fully field, Buckinghamshire, UK) with excitation at 427 nm and explained by the interactions ofthe enzyme with (a) MgPPi2- emission at 495 nm, both with a band width of 5 nm. The as the sole substrate and (b) Mg2' as an allosteric activator. standard reaction medium contained 3.0 jiM quinacrine, 50 mM KCI, 0.30 mM Na2EDTA, 150 mM mannitol, 25 mM BTP MATERIALS AND METHODS adjusted to pH 8.0 with Mes, tonoplast vesicles equivalent to approximately 200 ,ug protein per mL, and MgSO4 and Plant Material and Growth Conditions Na4PPi as indicated in the respective figures. The reaction Plants of Kalanchoe daigremontiana Hamet et Perrier de was initiated by the addition of Na4PPi. la Bathie were grown from leaf bulbils in a heated glasshouse All PPiase assays included 0.30 mM EDTA in the reaction under natural illumination supplemented by mercury-vapor medium, since this optimized the rates ofboth PPi hydrolysis lamps until 5 to 8 months old as described previously (37). and PPi-dependent H+ transport. This probably reflected an Plants were then transferred to a controlled-environment alleviation of PPiase activity from inhibition by micromolar room, where they were illuminated for 12 h daily at a photo- calcium concentrations (cf refs. 9 and 33) (L Skiera, D synthetic photon flux density of 300 ,umol m-2 s-' at mid- Sanders, personal communication), but the effect was not plant height provided by a combination of metal-halide flu- investigated further in the present study. orescent lamps and tungsten lamps. Air temperature was Protein concentrations were determined by the method of maintained at 25°C (light)/14°C (dark), with a relative humid- Bradford (3) using BSA as the standard. ity of approximately 35% (light)/70% (dark). Plants were maintained under these conditions for at least 7 d prior to Computations experiments. Concentrations of metal-ion complexes in the reaction media were calculated with the aid of the Fortran computer Isolation of Tonoplast Vesicles program Solcon, developed by Dr. D. C. S. White (University Tonoplast vesicles were isolated from homogenates of leaf of York, UK) and Dr. Y. E. Goldman (University of Penn- tissue of K. a of sylvania, Philadelphia, PA), using published association con- mesophyll daigremontiana by combination stants for all of the All differential and sucrose density gradient centrifugation as components reaction media. values described a microsomal were taken from Smith and Martell (28) except for the asso- previously (37). Briefly, supernatant, ciation constant for the formation ofMg2PPi, which was from obtained following differential centrifugation of mesophyll- Lambert and In at for 10 was over Watters (16). the computations, Solcon uses tissue homogenates 10,000g min, layered adjusted values ofthe association constants appropriate to the a 25% (w/v) sucrose cushion. This gradient was centrifuged the vesicles ionic strength and temperature of the reaction media. at 100,000g for 60 min, whereupon tonoplast Kinetic constants were determined by nonlinear regression banded at the interface between the supernatant and sucrose were removed and at analysis (37). cushion. Tonoplast vesicles pelleted Experiments were performed on average three times and 100,000g for 30 min, the final pellet being resuspended in a medium containing 150 mM mannitol, 1.0 mM DTT and 25 the graphical data present the results from a single character- BTP to 8.0 with Mes. All istic experiment. Values are expressed where appropriate as mM adjusted pH preparative steps means ± SE (n = number of samples). were performed at 4°C. Membranes were stored at -40°C and used within 1 week. RESULTS Assays Monovalent Cation Dependence of Tonoplast H+-PPiase Activity PPi hydrolysis was determined at 38°C from the release of Pi from Na4PPi as described earlier (27). The standard reac- To establish appropriate ionic conditions for investigating tion medium contained 50 mM KCI, 0.30 mM Na2EDTA, 1.0 the substrate kinetics ofthe tonoplast H+-PPiase ofKalanchoe mM Na2MoO4, 1.6 jiM gramicidin, 50 mM Tricine adjusted to daigremontiana, enzyme activity was first studied with respect pH 8.0 with Tris, tonoplast vesicles equivalent to approxi- to its monovalent cation dependence. The tonoplast H+- mately 5.0 ,ug protein per mL, and MgSO4 and Na4PPi as PPiase required K+ for maximal activity (Fig. 1), as found in indicated on the respective figures. Other differences in the other investigations (19, 24, 34, 35). In the absence of K+, composition of the reaction media are noted in the text. The PPi hydrolysis was 17 ± 4% (n = 5 experiments) of the reaction was initiated by the addition of Na4PPi and rates of maximum rate observed in the presence of K+ (Fig. IA), KINETICS OF TONOPLAST H+-PPiase 1 065
The apparent Ki (Na+) was identical to the Na+ concentration required for half-maximal activity in the absence of K+, suggesting that Na+ and K+ competed for the same site on v- the enzyme. Under the assay conditions developed for the
.01 present work, where the standard reaction medium contained
._ ._ 50 mM K+ and c 1 mM Na4PPi, the inhibition of PPi- 7F) 0 cn dependent H+ transport resulting from the presence of Na+ Co Q was less than 2%. E In the experiment of Figure 1, K+ concentration was varied
._ independently of Cl-, which was constant at 50 mm. Chloride itselfhas no direct effect on PPi hydrolysis, but it can stimulate v- PPi-dependent H+ transport into tonoplast vesicles by acting as a permeant anion, dissipating the inside-positive membrane potential generated by the H+-PPiase (19, 23, 35, 37). With a) the preparations used in this work, the Cl- concentration 03 giving half-maximal stimulation of PPi-dependent H+ trans- port was 15.2 ± 2.9 mm (n = 10 experiments), a value almost c identical to that for Cl- stimulation of ATP-dependent H+ c _.%100 transport in tonoplast vesicles of K. daigremontiana ( 12, a) 37). a Previous studies have yielded variable results concerning cCT a) 80 the dependence of the tonoplast H+-PPiase activity on K+, 0 0 with activities in the absence of K+ ranging from 3% (19) to L- 01)0) 56% (34) of maximum activities in the presence of K+. To 0) ) 60 test for the possible effects of other monovalent cations, PPi
o_ hydrolysis was also assayed in the presence ofdifferent 50 mM cation-Mes salts, with the following results (rates given in - 40 0) brackets as percentages of that in the presence of 50 mm K+): P* NH4+ [118] > Rb+ [101] ~ K+ [100] > Cs' [79] > Na+ [45] C.c ._ > Li' [38]. This sequence agrees with that found in other C investigations (19, 32, 34, 35). Additionally, however, we a found that some organic amines also supported appreciable 20 40 60 80 100 rates of PPi hydrolysis, specifically Tris+ [59] and tetrameth- ylammonium+ [48], as compared with BTP+ [17] in Fig. IA. K' concentration ( mM ) Thus, we conclude that the tonoplast H+-PPiase activity has Figure 1. Dependence of (A) PPi hydrolysis and (B) PPi-dependent an absolute requirement for a monovalent cation. The rela- H+ transport in tonoplast vesicles of K. daigremontiana on K+ con- tively high background activities observed in some studies in centration in the presence (0) and absence (0) of 50 mm Na-Mes. the absence of K+ may have simply reflected the ability of PPi hydrolysis was assayed as Pi release and H+-transport as the Tris+ ions present in those assay media to support PPi initial rate of quinacrine-fluorescence quenching as described in "Ma- hydrolysis. terials and Methods." Assays were performed in the presence of 0.5 mM BTP-PPi and 7.0 mm MgSO4, except that KCI was replaced by Dependence of H+-PPiase Activity on MgSO4 and 50 mM BTP-CI and K-Mes to give the indicated K+ concentrations. Na4PPi Concentrations The dependence of the tonoplast H+-PPiase activity of K. whereas PPi-dependent H+ transport (measured as the initial daigremontiana on Mg was next investigated by assaying the rate of quinacrine fluorescence quenching) showed an abso- enzyme at a range of fixed total PPi concentrations. Both PPi lute dependence on K+ (Fig. 1B). Both PPi hydrolysis and hydrolysis and PPi-dependent H+ transport increased sig- PPi-dependent H+ transport showed saturation kinetics with moidally with total Mg concentration (Fig. 2), with increasing respect to K+, with half-maximal activities being observed concentrations of total Mg being required for optimal H+- at K+ concentrations of 3.8 mm and 2.7 mm, respectively PPiase activity as the total PPi concentration was raised from (Fig. 1). 0.1 to 1.0 mM. In contrast to reports ofa substantial inhibition Sodium ions also supported PPi-dependent H+ transport of tonoplast H+-PPiase activity at supraoptimal total Mg (half-maximal activity being observed at 12 mM Na+), but at concentrations (4, 13, 17, 32, 35), little or no inhibition of low rates compared with K+: in the presence of 50 mm Na+, H+-PPiase activity was observed in the present experiments the rate was only 15 ± 2% (n = 5 experiments) of that with at total Mg concentrations between 5 and 10 mm (Fig. 2). 50 mM K'. When assayed in the presence of 2 mm Na+ (data When the converse experiment was performed and total not shown) and 50 mm Na+ (Fig. 1 B), the K+ concentrations PPi concentration was varied in the presence of a fixed, required for half-maximal stimulation of PPi-dependent H+ saturating total Mg concentration of 7.0 mM, PPi-dependent transport were increased to 8.9 mM and 17.1 mM, respectively. H+ transport showed Michaelis-Menten-type kinetics, with an 1 066 WHITE ET AL. Plant Physiol. Vol. 93,1990
At a saturating total Mg concentration (7.0 mM), it was not A possible to distinguish between MgPPi2-, A MgHPPi-, and 15 Mg2PPi as the true substrate for the enzyme, since all three _ ^% species increased in a similar manner with increasing total PPi concentration (Fig. 3). As a consequence, PPi-dependent H+ transport apparently showed Michaelis-Menten kinetics >16&_ ~~~~~~~~~~~~~withrespect to all three possible substrates, with predicted Km. )
Because free Mg2' appears to activate the tonoplast H+- I PPiase (13, 34, 35) and free PPi might inhibit it (34, 35), the c substrate dependence of the enzyme was further analyzed for 20 _ 100-a 10 0.a free PPi O results obtained in the presence ofexcess Mg2+ concentrations 4/ ~~~~~~~~C L- 0 (above 3 mM) and low concentrations of free PPi (i.e. PPi4- Mg2+ E + HPPi3 + H2PPi2 c 1.83 ltM). Under these conditions, E 115 _ . 7567 %._ substrate concentration alone would be expected to limit PPi ~c 0o hydrolysis and PPi-dependent H+ transport. Simple Michae- 10~'" - -- 5c E 1 lis-Menten kinetics were observed with respect to MgPPi2- Co 101.0 50 od 0) M._ concentration, but not Mg2PPi concentration, for both PPi 0 a- 0 hydrolysis (Fig. 7) and PPi-dependent H+ transport (not -6co 2- c.0O 0) shown), supporting the conclusion that MgPPi2- is the true ._n + 0 substrate of the tonoplast H+-PPiase. The kinetic parameters a- 2 with respect to MgPPi2- for PPi hydrolysis were Km = 16.8 ± I 2.3 AM and Vmax = 14.6 ± 0.4 Amol h-' mg protein-' (n = 24; 0 2 4 6 8 10 Fig. 7); for PPi-dependent H+ transport the corresponding Total Mg concentration (mM ) values were Km = 6.7 ± 2.6 AM and Vmax = 98.6 ± 4.6% fluorescence quench min-' mg protein-' (n = 29). Figure 5. Dependence of PPi hydrolysis and concentrations of ionic species in the assay medium on total Mg concentration, supplied as Activation of the Tonoplast H+-PPiase by Free Mg2+ MgSO4. Rates of PPi hydrolysis are replotted from Figure 2. Assays were performed in the presence of 0.1 mm Na4PPi and other com- Having identified the substrate of the tonoplast H+-PPiase ponents as indicated in "Materials and Methods." Free PPi concen- as MgPPi2-, it was possible to investigate the dependence of tration is the sum of PPi4-, HPPi3-, and H2PPi2- concentrations. The PPiase activity on free Mg2+ concentration. When plotted as concentration of MgHPPi- was below 1.30 mM. a percentage of the maximal activity at different MgPPi2- concentrations, PPi hydrolysis showed a sigmoidal depend- of the data was linear (Fig. 8, inset), giving a Hill coefficient ence on free Mg2> concentration, with half-maximal PPi (slope) of 2.52 ± 0.25 and an overall dissociation constant, hydrolysis occurring at a concentration of 1.1 mM (Fig. 8). Kd, for the Mg-enzyme complex given by the y-intercept as This result implies that free Mg2' acts as an allosteric activator -log Kd = 7.42 ± 0.78 (Fig. 8, inset). An equivalent analysis of the enzyme, with the enzyme showing positive cooperativ- of the activation of PPi-dependent H+ transport by free Mg2> ity with respect to Mg2> binding. A Hill plot transformation yielded a Hill coefficient of 2.74 ± 0.77 and -log Kd = 7.82 ± 2.46. The kinetics of Mg2> activation of the PPiase were unaffected by free PPi concentrations, which in the experi- ment of Fig. 8 varied between minimum values of <0.1 nM and maximum values of202 AiM for PPi4-, 633 AM for HPPi3-, 200 and 9 AM for H2PPi2-. 0 DISCUSSION c 150 C\ Ionic Requirements and Kinetics of the 1OO \ Tonoplast H+-PPiase The specific activity of the tonoplast H+-translocating 0- K. 50 PPiase from daigremontiana assayed in vitro is ofthe same order as that of the tonoplast H+-ATPase (12, 19, 37), sug- -x0~ 50_ _ gesting that the H+-PPiase could play a significant role in energizing vacuolar solute accumulation in vivo. However, the kinetic characteristics of the tonoplast H+-PPiase have C been difficult to resolve, and it is not yet clear how this 0 5 10 15 20 enzyme is regulated under physiological conditions. Time (min) Our results suggest that the tonoplast H+-PPiase possesses an absolute requirement for a monovalent cation for activity. Figure 4. Time course of PPi hydrolysis by the tonoplast H+-PPiase In K. daigremontiana the enzyme shows greatest activity in of K. daigremontiana. The assay medium (final volume 4 mL) initially contained 430 ,ug membrane protein, 7.0 mm MgSO4, 0.2 mm Na4PPi, the presence ofK+, which appears to be a characteristic feature and other components as indicated in "Materials and Methods." ofthe tonoplast H+-PPiase (19, 24, 32, 34, 35). However, PPi- Duplicate aliquots of 50 AL were removed at the times indicated. dependent H+ transport was more selective than PPi hydrol- Arrow indicates total PPi concentration at which the rate of PPi ysis for K+. The possibility that PPi hydrolysis in the absence hydrolysis (slope of tangent to the curve) was approximately of K+ is catalyzed by a different phosphatase cannot yet be half-maximal. eliminated (35), but we believe this K+-independent activity 1 068 WHITE ET AL. Plant Physiol. Vol. 93,1990
substrate for the enzyme. Johannes and Felle (13) concluded
I I I I I I~~~~~~~~~~~~~~~~~~~~~~ in their study that MgPPi>- and/or MgHPPi- is the substrate
1-00c._ for H+ transport by the tonoplast H+-PPiase of R. fluitans, -.2._ 20 0a-)2 but we believe the pH profile of the enzyme eliminates a M92 , _ c MgHPPi- as a possible substrate (see "Results"). In addi- 0 E _ tion, MgPPi2- appears to be the physiological substrate 15 co of the alkaline PPiases of microorganisms (7, 14, 15) and
o mitochondria (33). Having identified MgPPi>- as the substrate ofthe tonoplast E 10 _- 50 ._ H+-PPiase, the Mg-dependence of the enzyme was analyzed =- ol~~~~~~~~~~~~~~~ 25 c' for a range of substrate concentrations. This revealed that the 0 07 enzyme has an absolute requirement for free Mg2", showing
~0_ N _- CD no activity in its absence (Fig. 8). H+-PPiase activity increased sigmoidally with increasing Mg2" concentration (half-maxi- 25 X mal activity occurring at 1.1 mm free Mg"), suggesting that CL O I I I I I the enzyme was allosterically modulated and exhibited posi- CL tL 20 40 60 80 100 tive cooperativity with respect to Mg" binding. The linearity Total Mg concentration ( mM )
Figure 6. Dependence of PPi hydrolysis and concentrations of 20 Mg2PPi and MgPPi2- in the assay medium on total Mg concentrations I greater than 10 mm, supplied as MgSO4. Assays were performed in A the presence of 0.1 mm Na4PPi and other components as indicated in "Materials and Methods." The concentration of free PPi (the sum of PPi4-, HPPi3-, and H2PPi2- concentrations) was below 50 nm, 15 MgHPPi- below 510 nm, and Mg2+ concentration above 8 mM. AkL A 00** may simply reflect the ability of other monovalent cations 10 c commonly present in assay media, such as organic amines, to ._ support the hydrolytic activity of the tonoplast H+-PPiase. Tonoplast H+-PPiase activity showed sigmoidal kinetics 0. 5 with respect to total Mg concentration (Fig. 2), but hyperbolic 0) kinetics with respect to total PPi concentration (Fig. 3), as E MgPpi 2- found in other studies (5, 13, 19, 20, 23, 24, 32, 34, 35). These kinetics can be explained by the requirement of the enzyme 0: for free Mg2' as an activator in addition to a Mg/PPi complex E B as substrate. The tonoplast H+-PPiase of K. daigremontiana was not significantly inhibited by supraoptimal total Mg 0 concentrations up to 10 mm (Fig. 2), in contrast to H+-PPiase 15F activities in tonoplast preparations from roots ofAvena sativa ~0 (17, 35), internodal cells of Chara corallina (32), and thalli of L? 0* Ricciafluitans (13). These latter observations have led to the NMO0 suggestion that Mg2PPi is an inhibitor of the tonoplast H+- a. 101 PPiase (13), but this does not apparently apply to the enzyme 0 from K. daigremontiana. Indeed, little inhibition ofH+-PPiase 0 activity was observed in tonoplast fractions from roots of Zea 5 mays (5) or storage roots of Beta vulgaris (34) at high total 0I Mg concentrations. From measurements of PPi hydrolysis at increasing total 0 Mg2PP1 PPi concentrations (Fig. 3) or at low total Mg concentrations nII (Fig. 5), it was not possible to distinguish kinetically between 0 100 200 300 400 500 600 700 MgPPi2- and Mg2PPi as potential substrates of the tonoplast Concentration ( FM ) H+-PPiase. But in experiments performed at total Mg concen- Figure 7. Dependence of PPi hydrolysis on (A) MgPPi2- and (B) trations in excess of 10 mm (Fig. 6), it was clear that H+- Mg2PPi concentration in the presence of free Mg2+ concentrations in PPiase activity paralleled changes MgPPi2- rather than above 3 mm. Values are from experiments in which MgSO4 concen- Mg2PPi concentration. Rates of PPi hydrolysis showed Mi- tration was varied in the presence of 0.1 mM (U, O), 0.5 mm (0), and chaelis-Menten kinetics with respect to MgPPi2- (apparent 1.0 mm (A) Na4PPi (Figs. 2, 5, and 6). The curve was fitted by Km = 17 ,uM; Fig. 7), demonstrating that MgPPi2- is the true nonlinear regression analysis. KINETICS OF TONOPLAST H+-PPiase 1 069
140 , 1 at dawn; [22]). Nevertheless, it is possible to calculate an approximate cytosolic MgPPi2- concentration if the concen- tration of free Mg2+ is known. Little information is available - 120 A on cytosolic Mg2+ in plant cells, but a concentration of 0.4 2 * * * mM has been estimated in root tips of Vigna mungo using in 100 *-^ . * _ vivo 3'P-NMR spectroscopy (38). In animal cells, cytosolic A Mg2+2 concentrations have been determined in a large number of different tissues by various techniques and generally lie in 80 1.0 the range 0.4 to 2.0 mM (2, 21). Hence, assuming cytosolic concentrations of 0.4 mm for free and 200 AM for total 0 - Mg2+ *) 60f,0.5 ;- _ _ PPi, and that the contribution to total PPi of species other (0) kV loEo 0 > than H2PPi2, HPPi3-, PPi4, MgPPi2, MgHPPi-, and Mg2PPi is minimal, we can calculate the concentration of all a. 40 - >o 5 Z / - _ PPi complexes at a typical cytosolic pH of 7.5 and ionic /O + strength of 0.15 M. The major cytosolic PPi species is then 70 followed 6 2 20 _-1 - 1.0. . . _ MgPPi2- (1 AM), by Mg2PPi (1 ,uM), HPPi3- (1 -3.5 -3.0 -2.5 MM), PPi4 (2 Mm), H2PPi2 (0.31 Mm), and MgHPPi (0.25 log [Mg2+] Mm). The anticipated cytosolic MgPPi2- concentration is °O 4 6I 8 1 O therefore considerably higher than the apparent Km (MgPPi21) ofthe tonoplast H+-PPiase, suggesting that modulation ofH+- Free Mg2+ concentration ( mM ) PPiase activity through substrate limitation is unlikely. Inter- estingly, another important PPi-dependent enzyme, the cy- Figure E3. Dependence of PPi hydrolysis on free Mg2+ concentration. tosolic pyrophosphate:fructose-6-phosphate I-phosphotrans- Values are expressed as percentages of the predicted maximal ferase, has an almost identical Km for total PPi (7-16 Mm, activity at each Na4PPi concentration in the presence of saturating depending on Pi concentration) (29) to the tonoplast free Mg'+, calculated from the regression curve shown in Figure 7A. H+-PPiase. Inset: Hiill plot transformation of data for observed PPiase activities In contrast,He-PPiasetonoplast activity will be severely (v) betwteen 15 and 85% of the maximum (Vmax) plotted against log Ic invia freMg2tby +ifte ctosoty Mgll conen- molar fr(ee Mg2+ concentration. restricted in vivo by free if the cytosolic Mg2' concen- tration is 0.4 mM (38), since the enzyme would be operating at only 7% of its maximal velocity (Fig. 8). However, since of the ]Hill plot transformation is consistent with a Monod- the H+-PPiase is exceedingly sensitive to changes in free Mg2+ Wymar\I-.~Jn-ChangeauxLLIJLi 1~ mechanismL'~1L~ LL~in which the enzymeA would concentration in the range of 0.4 mM to 2.0 mM (cf. Fig. 8), exhibit simple concerted conformational changes upon the its activity could be strongly modulated by changes in cyto- binding of Mg2+. The calculated Hill coefficients of 2.5 for solic Mg2+. In root tips of V. mungo treated with azide, PPi hycirolysis and 2.7 for PPi-dependent H+ transport would, cytosolic free Mg2+ concentration was observed to increase on the assumption of a single binding site for Mg2+ per from 0.40 to 0.68 mM concomitant with a fall in the adenylate protomier, imply that the tonoplast H+-PPiase has at least (ATP/ADP) ratio (38). Also, cytosolic free Mg2+ concentra- tion increases from 0.85 mm to over 2.0 mm in heart tissue three iclenticallenticalMg2+-bindgMg+-binding subunits per holoenzyme. This drnishmastolAT lelsecnendMg2+ iS is cons,istent with recent data indicating that the tonoplast during ischemia as total ATP levels decline and g+i H+-PPiiasten withreposentdatmulindicatsuthat the tonoplt released from the MgATP2- complex (21). Thus, one role for ase is composed of multiple subunits (6, 26). the tonoplast H+-PPiase in plant cells may be to operate as an ancillary enzyme that is activated by increasing cytosolic Physiolllogical Implications Mg2+ concentration under conditions of limited ATP availa- Base(d on the kinetic properties of the tonoplast H+-PPiase bility. This could serve to maintain the proton-motive force describled above, we can assess how its activity might be across the tonoplast when H+-ATPase activity is restricted modulaited under physiological conditions in the cytosol. As and prevent loss of vacuolar solutes to the cytosol. noted tby Luttge (18), K+ is unlikely to regulate H+-PPiase Although a clearer picture is emerging of the kinetics of the activity in vivo, since the K50 for K+ stimulation is approxi- tonoplast H+-PPiase, further studies will be necessary to clarify mately 3 mM, while cytosolic K+ concentration is between 50 its exact physiological role in vacuolar solute accumulation and 1540 mm. Thus, changes in either substrate (MgPPi2-) or (25). Changes in tonoplast H+-PPiase activity and cytosolic free Mfg2+ concentrations are more likely to determine tono- PPi levels could be of considerable importance in the regula- plast H[+-PPiase activity. tion ofmetabolism (8, 31). Elucidation ofother characteristics Undcer many physiological conditions, cytosolic PPi con- of the tonoplast H+-PPiase such as its stoichiometry and centratiion appears to be relatively constant at about 200 gM directionality would therefore lead to a better understanding of its role in transport and metabolism. kO,(2R JVJ,2A\ ialLIIUUWIl.1thr~nIii thi-LI11 CtUbUULC;nhce-inte vnntVtIIUC klb bUUJC;%,riihiprt t,-LU 4abbUili"LIUllbnerinfne about subcellular compartmentation. In K. daigremontiana, ACKNOWLEDGMENTS however, there is a large fluctuation in total PPi concentration in the bulk mesophyll tissue during the CAM rhythm (from We are grateful to Dr. D. C. S. White and Dr. D. Sanders (Univer- 8 ,umol kg-' fresh weight at dusk to 88 Mmol kg-' fresh weight sity of York, UK) for kindly making available the computer program Plant Physiol. Vol. 93,1990 1070 WHITE ET AL.
Solcon. We also thank Dr. R. A. Leigh and Dr. A. J. Pope (AFRC emplifying the need for integration in ecophysiological work. Institute of Arable Crops Research, Rothamsted Experimental Sta- New Phytol 106: 593-629 of 19. Marquardt G, Luttge U (1987) Proton transporting enzymes at tion, UK) for communicating the results of their initial modeling the tonoplast of leaf cells of the CAM plant Kalanchoe daigre- the kinetics of the tonoplast H+-PPiase of oat roots, as well as Dr. P. montiana. II. The pyrophosphatase. J Plant Physiol 129: 269- A. Rea (University of Pennsylvania, Philadelphia) for helpful 286 discussions. 20. Mastowski P, Maskowska H (1987) Purification and some prop- erties of proton-translocating pyrophosphatase from microso- LITERATURE CITED mal vesicles of corn seedlings. Biochem Physiol Pflanz 182: 73-84 1. Bennett AB, O'Neill SD, Eilmann M, Spanswick RM (1985) H+- 21. 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