An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings

Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H⁺-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H⁺-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg²⁺-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H⁺-translocating PPase.     

Ethylene Biosynthesis-Inducing Xylanase : II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride

The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion.     

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.     

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

The effects of salt stress on polypeptides in membrane fractions from barley roots

Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [³⁵S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium-proton anti-porter in the tonoplast membrane.     

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (−196°C) of the membranes were irradiated with ⁶⁰Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.     

Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [³⁵S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [³⁵S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Translational modification of an 18 kilodalton polypeptide by spermidine in rice cell suspension cultures

When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-³H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis.     

Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H⁺ pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H⁺ pumping activities. N,N′-dicyclohexylcarbodiimide inhibited the ATPase strongly (I₅₀ = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4′-Stilbenedisulfonic acid affected both enzyme activities and their associated H⁺ pumping activities. This is in contrast to the H⁺-PPase of higher plants which is 4,4′-stilbenedisulfonic acid insensitive.     

Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase

The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Active Glucose Transport and Proton Pumping in Tonoplast Membrane of Zea mays L. Coleoptiles Are Inhibited by Anti-H-ATPase Antibodies

A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg²⁺-ATP dependent accumulation of 3-O-methyl-d-glucose (OMG) as determined by a membrane filtration technique using ¹⁴C-labeled substrate. OMG transport showed an apparently saturable component with a K_m of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H⁺-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes.     

A 36 Kilodalton Limiting-CO(2) Induced Polypeptide of Chlamydomonas Is Distinct from the 37 Kilodalton Periplasmic Carbonic Anhydrase

Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism, induced by limiting CO₂, which involves active transport and accumulation of inorganic carbon within the cell. Synthesis of several proteins is induced by limiting CO₂, but, of those, only periplasmic carbonic anhydrase has an identified function in the system. No proteins involved in active transport have yet been identified, but induced, membrane-associated polypeptides, such as the 36 kilodalton polypeptide focused on in this paper, would seem to be candidates for such involvement. The 36 kilodalton polypeptide was shown to be synthesized de novo upon transfer of cells to limiting CO₂. It was purified using SDS-PAGE and used to produce polyclonal antibodies. Antibodies were used to confirm the air-specific nature of the polypeptide, its strict association with membrane fractions, and the time course of its induction. Using the antibodies, a single, 36 kilodalton polypeptide was found to be specifically immunoprecipitated from in vitro translation products of poly(A⁺) RNA from cells only after exposure to limiting CO₂. The absence of translatable mRNA for this polypeptide in CO₂-enriched cells indicated that regulation occurs at the level of message abundance. The antibodies were also used to demonstrate the distinction between the limiting-CO₂ induced 36 kilodalton polypeptide and the similarly sized, limiting-CO₂ induced periplasmic carbonic anhydrase.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Molecular characterization of choline acetyltransferase from Drosophila melanogaster

Purified acetyl-CoA: choline O-acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster has been shown to contain two major polypeptides of 67 and 54 K Daltons. However, all enzyme activity is found in a single molecular weight form of approx 67 K Daltons as determined by sucrose gradient sedimentation and molecular exclusion chromatography. The latter showed both the 67 and 54 K Dalton polypeptides on polyacrylamide gel electrophoresis in sodium lauryl sulfate (10% acrylamide). Analysis of purified choline acetyltransferase on polyacrylamide gel electrophoresis in sodium lauryl sulfate (15% acrylamide) revealed the presence of an additional polypeptide at 13 K Daltons. Tryptic-peptide maps of the 67, 54 and 13 K Dalton components showed all three to be structurally related. In addition to several common tryptic peptides, the 13 K Dalton polypeptide contained three tryptic-peptides that were also found in the 67 K Dalton polypeptide, but were absent from the 54 K Dalton polypeptide. This evidence suggests that native Drosophila choline acetyltransferase may exist in two forms, one a single polypeptide chain with a molecular weight of 67 K Daltons and the other consisting of two noncovalently bound polypeptide chains with molecular weights of 54 and 13 K Daltons. The latter form is the major one isolated and may be generated by limited proteolysis of the single chain 67 K Dalton form.     

The Non-Ionic Detergent Brij 58 Conserves the Structure of the Tonoplast H+-ATPase of Mesembryanthemum crystallinum L. During Solubilization and Partial Purification

The effects of solubilization with Triton X-100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H⁺-ATPase of plants of Mesembryanthemum crystallinum performing C₃ photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H⁺-ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent K_M value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C₃ and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H⁺-ATPase due to the Triton X-100 treatment. After solubilization with Brij 58 the tonoplast H⁺-ATPase was partially purified by Superose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H⁺-ATPase was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H⁺-ATPase is important for enzyme structure and function.     

Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H⁺-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K_m value for MgATP²⁻ of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg²⁺ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca²⁺, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H⁺-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.     

Identification with a photoaffinity reagent of a tonoplast protein involved in vacuolar malate transport of Catharanthus roseus

The effect of N-(4-azido-salicylyl) aspartic acid (AzSA), a photolysable analogue of malate, was tested on the malate transport activity of tonoplast vesicles isolated from Catharanthus roseus cell suspension cultures. AzSA inhibited malate uptake in a competitive manner with a K_ti of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Photolysis of ¹²⁵I-labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after analysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate-binding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also protected the 37 kDa polypeptide from labelling. Citrate and, to a lesser extent, quinate afforded protection from labelling whilst other organic acids or aspartic acid (100 millimolar) did not. These photoprotection results are in good agreement with the data concerning the specificity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both in tonoplast vesicles and in isolated vacuoles. These results suggest the involvement of the 37 kDa polypeptide in vacuolar malate transport.