Fructose bisphosphatase of cestodes of <Emphasis Type="Italic">Bothriocephalus scorpii</Emphasis>

There have been studied the activity and properties of fructose bisphosphatase (FBPase) of Bothriocephalus scorpii parasitizing in pyloric appendages of the goby Myoxocephalus brandti. All subcellular fractions of B. scorpii (12 g/cytosol, 105 g/cytosol, mitochondria, and microsomes) have the FBPase activity. The enzyme has a high affinity to substrate and needs the presence of bivalent cations (Mg²⁺ or Mn²⁺). AMP inhibits the enzyme significantly. Action of various effectors has been studied. The monovalent (Na⁺, K⁺, Li⁺, NH₄ ⁺) and bivalent cations (Zn²⁺ and Cu²⁺) inhibit the enzyme activity.     

Activity and properties of fructose bisphosphatase of turbellarian Phagocata sibirica

Activity and properties of fructose bisphosphatase (FBPase) was studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. There was studied dependence of the FBPase reaction rate on the substrate concentration. For realization of the enzyme activity, the high affinity to substrate and presence of bivalent cations (Mg2+ or Mn2+) are necessary. The was studied the effect of various effectors as well as of monovalent (Na+, K+, Li+, and NH4+) and bivalent (Zn2+ and Cu2+) cations.     

ATPase in ripening strawberries

ATPase was found in 1000g, 13 000g and 80 00Og fractions from strawberry fruits. The optima pH for ATPase was the same (i.e. 6) for the 3 fractions, which also showed similar substrate specificity. However, the enzyme associated with the 80 000 g fraction showed the highest affinity for ATP and the maximum V_max/K_m value. As the fruit ripened, from the green to dark-red stage, ATPase activity in the 80 000 g fraction increased more than three times. The ATP content of the fruit pulp, which was high at the green stage, decreased as the fruit matured and ripened. Na⁺ and K⁺ slightly stimulated enzyme activity associated with the 1000 g,80 000 g and soluble fractions, whereas, Ca²⁺ and Mg²⁺ inhibited the enzyme activity in all fractions. However, the extent of inhibition due to divalent cations lessened as the fruit ripened.     

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1

To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate at 95°C and pH 8.0 with a half-life (t _1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg²⁺, while Li⁺ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.     

Schistosoma mansoni: effect of some cations on the proteolytic enzymes of cercariae

The effects of various salts on the proteolytic activity of extracts from Schistosoma mansoni cercariae were tested. Using an Azocoll substrate, stimulation (2 to 2.5-fold) of activity by the monovalent cations Na⁺ and K⁺ was demonstrated, with maximum stimulation at 20–40 mM concentrations. The divalent cations Mg²⁺ and Ca²⁺ stimulated proteolytic activity at low concentrations (between 0 and 10 mM) but inhibited activity at higher concentrations. The divalent cations Zn²⁺, Cu²⁺, Fe²⁺, and Co²⁺ were inhibitory even at very low concentrations. The results presented here are discussed in relation to previously described ion effects on cercarial infectivity.     

NaCl处理对西伯利亚白刺幼苗生长及离子平衡的影响

以1年生西伯利亚白刺水培幼苗为材料,研究了不同浓度NaCl(0、200、400mmol·L~(-1))处理对幼苗生长及不同器官(根、茎、叶)中Na~+、K~+、Ca~(2+)、Mg~(2+)的吸收、运输与分配的影响,探讨西伯利亚白刺的盐适应机制。结果表明:(1)200mmol·L~(-1) NaCl处理促进了西伯利亚白刺幼苗的生长及叶片肉质化程度,400mmol·L-1 NaCl处理显著抑制其生长。(2)随着NaCl处理浓度的升高,西伯利亚白刺幼苗根、茎、叶中Na~+含量显著增加,且叶中Na~+含量显著高于茎和根中;根系中K~+含量显著增加;根、茎、叶中Ca~(2+)、Mg~(2+)含量在200mmol·L~(-1) NaCl处理下保持平稳或上升,而在400mmol·L-1 NaCl处理下显著下降。(3)各器官中K~+/Na~+、Ca~(2+)/Na~+和Mg~(2+)/Na~+比值总体随NaCl处理浓度的升高呈下降趋势,且根部离子比值始终高于叶片和茎。(4)随着NaCl处理浓度的升高,西伯利亚白刺幼苗根-茎SK,Na显著下降,而根-茎SCa,Na、SMg,Na及茎-叶SK,Na、SCa,Na、SMg,Na逐渐提高。研究发现,西伯利亚白刺的盐适应机制主要是通过植株的补偿生长效应及叶片对Na~+的聚积作用实现的,同时也与根系对K~+的扣留及茎叶对K~+、Ca~(2+)、Mg~(2+)选择性运输能力增强有关。     

Effect of nutrients on the accumulation of glutamyl endopeptidase in the culture liquid ofBacillus intermedius 3–19

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid ofBacillus intermedius 3–19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, whereas inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca²⁺, Mg²⁺, Co²⁺) increased the production of glutamyl endopeptidase, but others (Zn²⁺, Fe²⁺, Cu²⁺) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase byB. intermedius 3–19 was elaborated.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

Characterization of cadmium removal by Rhodotorula sp. Y11

Experiments were conducted studying the removal of Cd²⁺ from water via biosorption using Rhodotorula sp. Y11. The effects of temperature and initial pH of the solution on biosorption were studied. Caustic and heat treatments showed different influences on the biosorption capacity, and the highest metal uptake value (19.38 mg g⁻¹) was obtained by boiling treated yeast cells. The presence of competing cations, such as Ag⁺, Cu²⁺, and Mg²⁺, except Na⁺ ions, significantly interfered with the metal uptake. Results indicate that the Langmuir model gave a better fit to the experimental data than the Freundlich equation. The q ₁₀ value was 11.38 mg g⁻¹ for Cd²⁺ uptake by Y11. Chemical modifications of the biomass demonstrated that carboxyl and amide groups play an important role in Cd²⁺ biosorption.     

Regulation of intracellular cyclic AMP levels in the white-rot fungus Phanerochaete chrysosporium during the onset of idiophasic metabolism

Adenylate cyclase activity in Phanerochaete chrysosporium was present in cell fractions sedimenting at 1,000xg, 15,000xg, and in the 150,000xg supernatant. A small amount of activity in the 1,000xg pellet could be solubilised by treatment with Triton X-100, and the enzyme in all fractions required an ATP-Mn²⁺ substrate. Adenylate cyclase activity in the 150,000xg pellet was low (0.003 nmol/mg protein·min) and may have resulted from contamination by other fractions. Highest adenylate cyclase specific activity (0.37 nmol/mg protein ·min) was recorded in the 150,000xg supernatant at the onset of idiophasic metabolism. During this growth phase, adenylate cyclase activity also increased in the 1,000xg pellet and was maximally 4.5-fold greater than that in primary phase cultures. No significant cAMP-phosphodiesterase activity could be detected during growht in any of the cell fractions or in the growth medium with either Mn²⁺, Mg²⁺, or Ca²⁺ as added cations. The extracellular cAMP concentration increased logarithmically during primary growth; however, in cultures in idiophasic metabolism cAMP levels remained constant and relatively low. We suggest that excretion into the medium is the principal means by which intracellular cAMP levels are decreased in P. chrysosporium.Abbreviation EB extraction buffer     

Purification and characterisation of alkaline cellulase produced by a novel isolate,<Emphasis Type="Italic"> Bacillus sphaericus</Emphasis> JS1

A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4--glucanase, E.C. 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5. CMCase was purified 192-fold by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23%. The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa. Using SDS-PAGE a single band is found at 42 kDa. This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases. The activity of the enzyme was significantly inhibited by bivalent cations (Fe³⁺ and Hg²⁺, 1.0 mM each) and activated by Co²⁺, K⁺ and Na⁺. The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose. Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.     

Effeet of Metal Ions on Activity of Exopectic Acid Transeliminase of Erwinia sp.

Contrary to the exopectic acid transeliminase of Clostridium multifermentans, that of Erwinia sp. was activated strongly by Na⁺ and to a much less extent by Ca²⁺. K⁺ had a small stimulating effect on the enzyme activity. Mn²⁺ and Co²⁺, like Ca²⁺, activated the enzyme weakly. Ba²⁺ and Mg²⁺ showed no and a slight inhibitory effect, respectively, on the activity.

An almost total loss of activity was caused by the addition of EDTA to the reaction mixture. In the presence of Na⁺ the enzyme activity was restored by addition of divalent cations. Individual monovalent cations or each of the divalent cations was ineffective in restoring the activity.     

An Energy-Barrier Model for the Permeation of Monovalent and Divalent Cations Through the Maxi Cation Channel in the Plasma Membrane of Rye Roots

The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K⁺, Na⁺, Ca²⁺ and Ba²⁺ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model), which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary conductance vs. activity relationships for K⁺, Na⁺ and Ca²⁺, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative cation permeabilities with solution composition, and the complex effects of Ba²⁺ and Ca²⁺ on K⁺ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input to electrocoupling models allowing the relationships between membrane voltage, Ca²⁺ influx and Ca²⁺ signaling to be studied theoretically. Received: 29 April 1998/Revised: 20 November 1998     

Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes

These experiments examined effects of several ligands on the K⁺ p-nitrophenylphosphatase activity of the (Na⁺,K⁺)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K⁺-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K⁺ was approximately 75% less than that observed when K⁺ was included in the incubation medium. The response to the H⁺ concentrations was different: K⁺-independent activity showed a pH optimum around 6.5–7.0, while the K⁺-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K⁺-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K⁺-independent activity. Na⁺ did not affect K⁺-independent activity at all, while the same ligand concentration inhibited sharply the K⁺-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K⁺-dependent activity was stimulated by Mg²⁺ with low affinity (millimolar range), and 3 mM Mg²⁺ produced a slight stimulation of the activity in absence of K⁺, which could be interpreted as Mg²⁺ occupying the K⁺ sites. Ca²⁺ had no appreciable effect on the activity in the absence of K⁺. However, in the presence of K⁺ a sharp inhibition was found with all Ca²⁺ concentrations studied. ATP (0.5 mM) did not affect the K⁺-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K⁺, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used p-NPP p-nitrophenylphosphate - p-NPPase rho-nitrophenylphosphatase activity     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

The spatial distribution of cations in nematocytes of Hydra vulgaris

The spatial distribution of cations was assayed qualitatively and quantitatively in tentacular nematocytes of Hydra vulgaris in a scanning transmission electron microscope by means of x-ray microanalysis performed on 100 nm thick freeze-dried cryosections. The matrix of undischarged cysts (stenoteles, desmonemes and isorhizas) was found to contain mainly K⁺. In isolated nematocysts of Hydra the intracapsular potassium can be readily substituted by practically any other mono- and divalent cation (Na⁺, NH₄ ⁺, Mn²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺) all, except Fe²⁺, without impairing the ability of the cyst to respond to the chemical triggering with dithioerythritol or proteases. Monovalent cations increase the osmotically generated intracapsular pressure compared to divalent ions.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.