An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

ATPase activity associated with vacuoles and tonoplast vesicles isolated from the CAM plant, Kalanchoë daigremontiana

Intact vacuoles were isolated from leaves of the CAM plant, Kalanchoë daigremontiana Hamet et Perr. Both ATPase and acid phosphatase activities were found in the vacuoles. Purified tonoplast vesicles showed only ATPase activity with a pH optimum of 8.0. This activity was Mg²⁺-dependent and KCI or NaCI caused a further stimulation. N,N'-dicyclohexylcarbodiimide, diethylstilbestrol and quercetin inhibited the ATPase almost completely at concentrations well below 1 m M. NaVo₃, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, oligomycin and NaN₃ had little or no effect. Carbonyl cyanide m -chlorophenylhydrazone stimulated the ATPase about 40% at 5 × 10⁻⁴ M. The K_m for ATP was found to be 0.55 m M. These results indicate that the ATPase found in the tonoplast membrane of Kalanchoë daigremontiana is qualitatively similar to that of other plant species.     

Partial purification and properties of a 36-kDa 1-aminocyclopropane-1-carboxylate N-malonyltransferase from mung bean

A 36-kDa 1-aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase, which converts the ethylene precursor ACC into the conjugated derivative malonyl-ACC (MACC), has been isolated from etiolated mung bean ( Vigna radiata ) hypocotyls, and partially purified in a four-step procedure. The enzyme is stimulated about 7-fold by 100 m M K⁺ salts or 0.5 m M Co²⁺ salts, and is inhibited competitively by D-phenylalanine (K_i= 1.3 m M ) and non competitively by CoASH (0.3 m M ). Beside malonyl-CoA, it is capable to use succinyl-CoA as an acyl donor. The 36-kDa enzyme described here exhibits a lower optimum temperature (40°C) and a 7- or 3-fold lower apparent K_m for ACC (68 μ M ) and malonyl-CoA (74 μ M ), respectively, when compared with its 55 kDa isoform already isolated from the same plant material. This data support the idea that several isoforms of ACC N-malonyltransferase exist in plants. These isoforms may play a differential role in regulating the availability of ACC, and consequently the rate of ethylene production, as well as detoxifying cells from D-amino acids.     

Porphyrinogenesis in maize as affected by bidentate chelators of iron

Intracellular compartmentation of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) in wheat ( Triticum aestivum L. cv. Kanzler) and barley ( Hordeum vulgare L. cv. Gerbel) leaves was studied using different methods: first, the isolation of intact vacuoles from protoplasts and, second, a non-aqueous fractionation procedure. The two methods gave similar results. ACC concentrations were similar in the extravacuolar space and in the vacuole, whereas MACC was accumulated in the vacuolar space. Transport studies revealed that no specific carrier for ACC exists at the tonoplast. MACC transfer across the tonoplast was enhanced by 120% in the presence of ATP. MACC competitively inhibited malate transport into the vacuole indicating that the same transfer system catalyzes the transfer of the two dicarboxylates.
It is concluded that malonylation of ACC is not a prerequisite for the transport of ACC through the tonoplast.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Uptake of salicylic acid 2-O-beta-D-glucose into soybean tonoplast vesicles by an ATP-binding cassette transporter-type mechanism

In soybean ( Glycine max L.), salicylic acid (SA) is converted primarily to SA 2- O - β - d -glucose (SAG) in the cytoplasm and then accumulates exclusively in the vacuole. However, the mechanism involved in the vacuolar transport of SAG has not been investigated. The vacuolar transport of SAG was characterized by measuring the uptake of [¹⁴C]SAG into tonoplast vesicles isolated from etiolated soybean hypocotyls. The uptake of SAG was stimulated about six-fold when MgATP was included in the assay media. In contrast, the uptake of SA was only stimulated 1.25-fold by the addition of MgATP and was 2.2-fold less than the uptake of SAG providing an indication that the vacuolar uptake of SA is promoted by glucosylation. The ATP-dependent uptake of SAG was inhibited by increasing concentrations of vanadate (64% inhibition in the presence of 500 μ M ) but was not very sensitive to inhibition by bafilomycin A₁ (a specific inhibitor of vacuolar H⁺-ATPase; EC 3.6.1.3), and dissipation of the transtonoplast H⁺-electrochemical gradient. The SAG uptake exhibited Michaelis–Menten-type saturation kinetics with a K _m value of 90 μ M for SAG. SAG uptake was inhibited 60% by β ‐estradiol 17-( β - d -glucuronide), but glutathione conjugates and uncharged glucose conjugates were only slightly inhibitory. Based on the characteristics of SAG uptake into soybean tonoplast vesicles it is likely that this uptake occurs through an ATP-binding cassette transporter-type mechanism. However, this vacuolar uptake mechanism is not universal since the uptake of SAG by red beet ( Beta vulgaris L) tonoplast vesicles appears to involve an H⁺-antiport mechanism.     

Electrogenic proton translocation by the ATPase of sugarcane vacuoles

Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation.     

Measurement of intracellular pH in maize root tissue with α-methyl fluorinated alanines and in-vivo 19F NMR spectroscopy

Methods for the simultaneous measurement of vacuolar and cytoplasmic pH in plant tissues currently have significant limitations. This study demonstrates the usefulness of methyl difluoro alanine (F₂ALA) and methyl trifluoro alanine (F₃ALA) with in-vivo ¹⁹F NMR spectroscopy to measure vacuolar and cytoplasmic pH in maize root tissue. The pH dependence of the chemical shift of F₂ALA and F₃ALA is greater than either the commonly used ³¹P NMR signal of inorganic phosphate or the ¹³C NMR signals of trans-aconitic acid, which is also found in some plant cells. F₂ALA and F₃ALA were also able to detect changes over a greater range of pH. When maize root tissue was incubated in the presence of 0.35 m m F₂ALA or F₃ALA, these accumulated to significant concentrations in two compartments of different pH with no significant effect on growth rate of root tips. The time course of accumulation and the pH of the two compartments were consistent with one being the cytoplasm and the other the vacuole. The chemical shift of both C₂ of trans-aconitic acid and vacuolar F₃ALA indicated that the mean vacuolar pH of maize root cells was 4.6 and that the pH gradient across the tonoplast membrane was about 2.8 units. Under a variety of conditions, there was considerable heterogeneity in the pH of the vacuoles in maize root tissue as indicated by the peak width of the signal from F₃ALA. The significance of these values is discussed in terms of the bioenergetics of proton transport across the tonoplast membrane in vivo.     

2H+:1 malate2− stoichiometry during Crassulacean Acid Metabolism is unaffected by lipophilic cations

Abstract. Lipophilic cations inhibit nocturnal malic acid accumulation in leaf cells of the Crassulacean Acid Metabolism plant Kalanchoë tubiflora . perhaps by interacting directly or indirectly with active malic acid transport into the vacuoles. Lipophilic cations do not affect passive efflux of malic acid from the vacuoles. Membrane potentials are depolarized, oxygen uptake is stimulated by lipophilic cations and there may also be stomatal responses. Thus it is striking that lipophilic cations do not alter the stoichiometry of 2 titratable H : 1 enzymatically-determined malate²⁻ during diurnal malic acid oscillations of Crassulacean Acid Metabolism in Kalanchoë . This suggests that coupling between protons and malate during transport into the vacuole must be tight. Transport as undissociated acid is unlikely because the dissociation equilibrium in the cytoplasm is largely on the side of malate²⁻. These results appear to suggest an intimate molecular interaction between a proton pump and a presumed malate²⁻ translocator at the tonoplast of leaf cells with Crassulacean Acid Metabolism.     

The lysolipid sphingosine modulates pyrophosphatase activity in tonoplast vesicles and isolated vacuoles from a heterotrophic cell suspension culture of Chenopodium rubrum

The proton pumping activity of the tonoplast (vacuolar membrane) H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (K_a= 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent K_m= 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase.     

Regulation by CO2 of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climacteric fruits

Cheverry, J. L., Sy, M. O., Pouliquen, J. and Marcellin, P. 1988. Regulation by CO₂ of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climateric fruits. - Physiol. Plant. 72: 535–540.
A high CO₂ concentration (20%) at 20°C rapidly and strongly inhibited the development of the climacteric ethylene burst in apple ( Malus domestica Borkh. cv. Granny Smith) and avocado ( Persea americana Mill. cv. Fuerte) fruits and did not change 1-aminocyclopropane-l-carboxylic acid (ACC) content. Treatment with 20% CO₂ markedly decreased ACC-dependent ethylene biosynthesis at 20°C in climacteric pericarp tissues. It is suggested, therefore, that high CO₂ levels inhibit conversion of ACC to ethylene.
Synthesis of the ethylene forming enzyme (EFE) was enhanced when intact preclimacteric apples or early climacteric avocados were pretreated for 40 h with 10 μ11^-1 ethylene. When CO₂ (20%) and ethylene were both applied, a reduced stimulatory effect of ethylene on EFE synthesis was observed. A high CO₂ concentration enhanced EFE acivity in excised tissues of apples and avocados incubated with ACC (2 m M ) and cycloheximide (1 m M ) or 2–5-norbornadiene (5 ml 1^-1). In the autocatalytic process, 20% CO₂ antagonized the stimulation of EFE synthesis by ethylene, but promoted EFE activity.     

Stereoselectivity and structural determinants in molecular recognition by the ACC transport system in isolated maize mesophyll vacuoles

The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is actively transported across the tonoplast of plant cells, impacting cellular compartmentation of ACC and ethylene biosynthesis. In the present study, the effects of ACC and amino acid analogs on ACC uptake into isolated maize (Zea mays L. cv. Golden Cross Bantam) mesophyll vacuoles were investigated to identify the stereospecific and structural features that are important in molecular recognition by the ACC transport system. Of the four stereoisomers of l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC), (1S, 2R)-(–)-AEC having a configuration corresponding to an L-amino acid was the preferred substrate for the ACC transport system, competitively inhibiting ACC transport with a Ki of 18 μM. Of 11 neutral amino acid stereoisomers, L-isomers were stronger inhibitors of ACC transport than corresponding D-isomers. Neutral L-amino acids with nonpolar side chains generally were more inhibitory than those with polar side chains, whereas several cationic and anionic L-amino acids were ineffective antagonists of ACC transport. These observations suggest that the ACC transport system is stereospecific for relatively nonpolar, neutral L-amino acids. This conclusion was supported by the observation that group additions, substitutions, or deletions at the carboxyl. α-amino and the Pro- (R) methylene or hydrogen moieties (analogous to D-amino acids) of ACC and other neutral amino acids and analogs essentially eliminated transport inhibition. In contrast, L-amino acid analogs with variable substitutions at the distal end of the molecule remained antagonists. The relative activity of analogs was influenced by the length and degree of unsaturation of the side chain and by the location of side chain branching. Increasing the ring size of ACC analogs reduced antagonism whereas incorporating the α-amino group into the ring structure as an L-amino acid increased antagonism. The kinetics of L-methoxyvinylglycine, L-methionine. p-nitro-L-phenylalanine and 1-aminocyclobutane-l-carboxylic acid were competitive with Ki values of 3, 13, 16 and 19 μM, respectively. These results indicate that the ACC transport system can be classifie as a neutral L-amino acid carrier having a relatively high affinity for ACC and other nonpolar amino acids. The results also suggest that the carrier interacts with the carboxyl, α-amino and Pro-(R) groups and with other less restricted side chain substituents of substrate amino acids.     

Synergistic effect of 1-aminocyclopropane-1-carboxylic acid and ethylene during senescence of isolated carnation petals

The effects of ethylene (C₂H₄), (2-chloroethyl)phosphonic acid (ethefon) and 1-aminocyclopropane-1-carboxylic acid (ACC) on senescence of isolated intact petals and of upper petal parts of carnation flowers ( Dianthus caryophyllus L. cv. White Sim) were investigated.
Isolated upper petal parts did not respond to treatment with ethefon or ACC. These tissues did, however, show severe wilting in intact petals that were treated with ethefon or ACC. When isolated upper petal parts were simultaneously treated with ACC and ethefon or ACC and ethylene, a marked synergistic effect on senescence was found. Treatment of isolated petals with radiolabeled ACC led to the accumulation of radiolabeled ACC and N-malonyl-ACC (MACC) in the upper parts. The formation of ethylene and the malonylation of ACC were inhibited by pretreatment of the flower with the inhibitor of ethylene action, silver thiosulphate (STS), which indicates that both were induced by endogenously produced ethylene. Treatment of isolated upper parts with ACC slightly increased their ethylene production. However, when these petal parts were simultaneously treated with ethylene and ACC, the conversion of ACC to ethylene was markedly stimulated.
The results indicate that, in intact petals, ethylene may be translocated from the basal to the upper part where it stimulates the activity of the ethylene-forming enzyme (EFE), thereby making the tissue receptive to ACC.
In addition, it was found that upon incubation of petal portions in radiolabeled ACC, both the petal tissue and the incubation solutions produced radiolabeled carbon dioxide. This was shown to be due to microorganisms that were able to metabolize the carbon atoms in the 2 and 3 position of ACC into carbon dioxide.     

The role of Mg2+ in proton transport by the tonoplast pyrophosphatase in Riccia fluitans vacuoles

The Mg²⁺-dependent activity of the tonoplast pyrophosphatase (PPase) was investigated by measuring proton transport and by using the acridine orange technique on intact vacuoles of the aquatic liverwort Riccia fluitans L. In solutions with both Mg²⁺ and pyrophosphate present, a number of complexes are formed, which could all influence the enzymatic and hence the transport activity of the PPase. Therefore, the individual concentrations of these complexes were calculated and their contributions to proton transport across the tonoplast were tested. From these experiments we conclude that Mg²⁺ has three different roles: (i) Mg²⁺ stimulates transport activity of the PPase. (ii) Mg₂PP_i inhibits PPase-mediated H⁺ transport, (iii) MgPP_i* (= MgPP_i^2-+ MgHPP_i^-) is the substrate with an apparent K_1/2= 5–10 μM, with no discrimination between MgPP_i^2- and MgHPP_i^-.     

Tonoplast stability and survival of isolated vacuoles in different buffers

The mechanism of sucrose transport into vacuoles isolated from leaf tissue has been studied only in barley (Hordeum vulgare) mesophyll cells. In this tissue, sucrose transport was reported to be a facilitated diffusion. We have observed a facilitated diffusion of sucrose into vacuoles isolated from this tissue. However, no pH dependence was observed. Evidence is presented indicating that the pH dependence of sucrose uptake into vacuoles may be an artifact, reflecting tonoplast instability and survival of isolated vacuoles in different buffers. Apparently vacuoles do not withstand exposure to some commonly used buffers.     

Effects of propylene and oxygen on the ethylene-producing system of apples

Hypobaric conditions and treatments with ethylene and the ethylene analogue propylene were used to investigate effects of oxygen and elhylene on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity and ethylene production of apples ( Malus sylveslris Mill. cv. Golden Delicious). Prcclimacteric apples were stored in air at 6.6 kPa (reduced pressure); 6.6 kPa ventilated with pure O₂; 6.6 kPa ventilated with 2600 μl 1⁻¹ C₂H₄; and in air at 101.3 kPa (atmospheric pressure) for 4 months at 4°C. No ACC synthase activity was detectable in apples stored at 6.6 kPa, whereas ACC synthase activity was induced in apples stored at 6.6 kPa and ventilated with either O₂ or C₂H₄. In a further experiment, preclimacteric apples were stored for 14 days either in air at 20 kPa or at 20 kPa ventilated with pure O₂. Both treatments were supplied with 58 500 μl 1⁻¹ propylene from day 0 to day 9 or from day 9 to day 12. Ethylene production of apples treated with propylene from day 0 to day 9 increased earlier than ethylene production of untreated apples. Propylene treatment from day 9 to day 12 did not stimulate ethylene production. Ethylene and propylene induced and stimulated extractable ACC synthase activity and ACC formation of apples. Oxygen enhanced this effect. The results also suggest inhibition of in vivo ACC synthase activity by propylene.     

Ethylene formation in plant mitochondria. Dependence on the transport of 1-aminocyclopropane-1-carboxylic acid across the membrane

The mitochondrial fraction isolated from plumular hooks of etiolated pea seedlings ( Pisum sativum L. cv. Kelvedon Wonder) displayed a ten-fold higher rate of ethylene formation from 1-aminocyclopropane-1-carboxylic acid [ACC; 3.2 nmol C₂H₄ (mg protein ⁻¹)h⁻¹], than the tissue from which it was isolated. When the ionophores valinomycin or nigericin were added, a 60- to 70-fold increase in activity in intact mitochondria over the activity in plumular hooks was obtained for ethylene formation under the same conditions, and a 20-fold increase was obtained upon addition of gramicidin. The addition of ionophores did not affect the rate of ethylene formation in submitochondrial particles (55% inside-out as determined by cytochrome oxidase latency) which already exhibited a 2–3-fold higher specific activity than intact mitochondria. Low concentrations of the detergents cholate and deoxycholate increased mitochondrial ethylene formation activity and had no effect on the rate of the reaction in submitochondrial particles. These results support the suggestion that ACC conversion to ethylene is associated with the inner side of the inner mitochondrial membrane and that transport across the intact mitochondrial membrane is rate-limiting in the reaction.     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

Changes in spectral properties of ageing and senescing maize and sunflower leaves

Activity and biochemical characteristic of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase from pear ( Pyrus communis cv. Blanquilla) was determined. The enzyme showed a low K_m (57.5 μM) for ACC and was dependent on O₂ (K_m 0.44% in atmosphere). It had an absolute requirement for Fe²⁺, ascorbate and CO₂ and was inhibited by α-aminoisobutyric acid (AIB: K₁ 4.2 m M ) and cobalt. ACC oxidase has an optimum pH of 6.7 and temperature maxima at 28 and 38°C and it is concluded that the activity of ACC oxidase from pear resembles authentic in vivo activity.     

Effects of pH on proton transport by vacuolar pumps from maize roots

Protons pumps of the tonoplast may be involved in the regulation of cytosolic pH, but the effects of pH on the coupled activities of these transporters are poorly understood. The effects of pH on the activities of the H⁺-translocating pyrophosphatase (PP_iase) and vacuolar-type H⁺-translocating adenosine triphosphatase (H⁺-ATPase) from maize ( Zea mays L. cv. FRB 73) root membranes were assessed by model that simultaneously considers proton transport by the pump and those processes that reduce net transport. The addition of either pyrophosphate or ATP to either microsomal or tonoplast membranes generated a pH gradient. The pH gradient generated in the presence of both substrates was not the sum of the gradients produced by the two substrates added separately. When membranes were separated by sucrose density gradient centrifugation, pyrophosphate (PP_i)-dependent proton transport was associated with light density membranes having tonoplast H⁺-ATPase activity. These results indicate that some portion of the PP_iase was located on the same membrane system as the tonoplast ATPase; however, tonoplast vesicles may be heterogeneous, differing slightly in the ratio of ATP- to PP_i-dependent transport. Proton transport by both the PP_iase and ATPase had maximal activity at pH 7.0 to 8.0 Decreases in proton transport by the ATPase at pH above the optimum were associated with increases in the processes that reduce net transport. Such an association was not observed at pH values below the optimum. These results are discussed in terms of in situ regulation of cytoplasmic pH by the two pumps.