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Furfural is an aromatic aldehyde, classified as hazardous material that cause environmental impact, especially harmful for humans if consumed. Furfural removal in water is necessary to overcome the negative impact. Furfural removal can be carried out using adsorption of activated carbon from durian shell. This study determines contact time and reaction kinetic using modified activated carbon which carried out in 7 stages: preparation of furfural main solution 1000 ppm, furfural work solution, determining furfural standard curve, adsorption time, best mass and adsorption capacity of activated carbon, isotherm adsorption and kinetics. The parameters measured are contact time, capacity, furfural adsorption kinetics. Filtrate testing is carried out using UV-Spectrophotometry. Results that obtained in this study are best contact time at 75 minutes with reaction order; reaction contant (k); linear regression (R2) are 1.5; 3.966 × 10-5 (mg/g)/s; 0.9451 respectively. The isotherm adsorption model is Langmuir Isotherm.

Phytochelatin (PC) synthases are γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (γ-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes γ-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes γ-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent γ-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary γ-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary γ-Glu-Cys acylation.

γ-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low Km values (0.5–2 μm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with ∼50 and ∼10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16–60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.

Phytochelatin (PC) synthase has been assumed to be a γ-glutamylcysteine dipeptidyl transpeptidase (EC 2.3.2.15) and, more recently, as exemplified by analyses of the immunopurified recombinant enzyme from Arabidopsis thaliana (AtPCS1-FLAG), has been shown to catalyze a PC synthetic reaction with kinetics that approximates a bisubstrate-substituted enzyme mechanism in which millimolar concentrations of free GSH and micromolar concentrations of heavy metal·GSH thiolates (e.g. cadmium·GS2) or millimolar concentrations of S-alkylglutathiones serve as cosubstrates. Here, we show, by direct analyses of the stoichiometry of AtPCS1-FLAG-catalyzed PC synthesis, the kinetics and stoichiometry of acylation of the enzyme and release of free glycine from γ-Glu-Cys donors, and the effects of the Cys-to-Ser or -Ala and Ser-to-Ala substitution of conserved residues in the catalytic N-terminal half of the enzyme, that PC synthase is indeed a dipeptidyltransferase that undergoes γ-Glu-Cys acylation at two sites during catalysis, one of which, in accord with a cysteine protease model, likely corresponds to or is at least tightly coupled with Cys56. The identity of the second site of enzyme modification remains to be determined, but it is distinguishable from the first Cys56-dependent site, which is amenable to γ-Glu-Cys acylation by free GSH, because its acylation not only depends on the provision of Cd2+ or GSH with a blocked, S-alkylated thiol group, but is also necessary for net PC synthesis. We conclude that des-Gly-PCs are not generated as an immediate by-product, but rather that the enzyme catalyzes a dipeptidyl transfer reaction in which some of the energy liberated upon cleavage of the Cys–Gly bonds of the γ-Glu-Cys donors in the first phase of the catalytic cycle is conserved through the formation of a two site-substituted γ-Glu-Cys acyl-enzyme intermediate whose hydrolysis provides the energy required for the formation of the new peptide bond required for the extension of PC chain length by one γ-Glu-Cys repeat per catalytic cycle.

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

The dependence of phytochelatin synthase (γ-glutamylcysteine dipeptidyltranspeptidase (PCS), EC 2.3.2.15) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana(AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd2+- and Zn2+-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g.Cd·GS2 or Zn·GS2) and free glutathione act as γ-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both γ-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.

Plant vacuolar H+-translocating inorganic pyrophosphatases (V-PPases; EC 3.6.1.1) have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVP1 (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad Sci USA 89: 1775–1779) from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K+-stimulated H+ translocation. Here we describe the molecular and biochemical characterization of AVP2 (accession no. AF182813), a sequence-divergent (36% identical) K+-insensitive, Ca2+-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H+ translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPases from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AVP1, is but one representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum.

Phytochelatins, a class of posttranslationally synthesized peptides, play a pivotal role in heavy metal, primarily Cd 2+ , tolerance in plants and fungi by chelating these substances and decreasing their free concentrations. Derived from glutathione and related thiols by the action of γ-glutamylcysteine dipeptidyl transpeptidases (phytochelatin synthases; EC 2.3.2.15 ), phytochelatins consist of repeating units of γ-glutamylcysteine followed by a C-terminal Gly, Ser, or β-Ala residue [poly-(γ-Glu-Cys) n -Xaa]. Here we report the suppression cloning of a cDNA ( AtPCS1 ) from Arabidopsis thaliana encoding a 55-kDa soluble protein that enhances heavy-metal tolerance and elicits Cd 2+ -activated phytochelatin accumulation when expressed in Saccharomyces cerevisiae . On the basis of these properties and the sufficiency of immunoaffinity-purified epitope-tagged AtPCS1 polypeptide for high rates of Cd 2+ -activated phytochelatin synthesis from glutathione in vitro , AtPCS1 is concluded to encode the enzyme phytochelatin synthase.

On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane alpha-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thaliana were singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229 (227-245) of the V-PPase and the N,N'-dicyclohexylcarbodiimide (DCCD)-binding transmembrane alpha-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y. , and Strid, A. (1993) Plant Cell Physiol. 34, 375-378). Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae. Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type. The one class II mutant obtained (E427Q) was preferentially impaired for H+ translocation over PPi hydrolysis but retained sensitivity to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+ translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident. The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase. The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation. Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding. The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+ translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides.

We have studied the uptake of [3H]-medicarpin and its glutathione conjugate(s) into vacuolar membrane vesicles from etiolated hypocotyls of mung bean (Vigna radiata). Unconjugated medicarpin is taken up at a low rate in the presence or absence of MgATP. However, [3H]-medicarpin-glutathione conjugate(s), prepared by incubation of medicarpin with a total maize glutathione S-transferase preparation, is taken up more than four-fold faster than medicarpin in the presence of MgATP, and this uptake is MgATP-dependent. Uptake of medicarpin-glutathione was not significantly inhibited by the ionophore gramicidin-D, but was strongly inhibited by vanadate and the alternative transport substrate S-(2,4-dinitrophenyl) glutathione. Our results demonstrate, in a model system, the potential utilization of the high affinity, high capacity, uncoupler-insensitive glutathione conjugate pump for the vacuolar transport of an isoflavonoid phytoalexin.

A characteristic feature of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase) of plant cells is its high sensitivity to irreversible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl reagents. Previous investigations in this laboratory have demonstrated that the primary site for substrate-protectable covalent modification of the V-PPase by 14C-labeled NEM maps to a single Mr 14,000 V8 protease fragment (V814K) (Zhen, R.-G., Kim, E. J., and Rea, P. A.(1994) J. Biol. Chem. 269, 23342-23350). Here, we describe site-directed mutagenesis of the cDNA encoding the V-PPase from Arabidopsis thaliana, its heterologous expression in Saccharomyces cerevisiae and single substitution of all 9 conserved Cys residues to either Ser or Ala. In all cases, except one, Cys mutagenesis exerts little or no effect on either the catalytic activity or susceptibility of the enzyme to inhibition by NEM. By contrast, and in complete agreement with the results of peptide mapping experiments, substitution of Cys634, the sole conserved cysteine residue encompassed by V814K, with Ser or Ala generates enzyme that is insensitive to NEM but active in both PPi hydrolysis and PPi-dependent H+ translocation. The specific requirement for Cys634 for inhibition by NEM and the dispensability of all of the conserved Cys residues, including Cys634, for V-PPase function indicate that the inhibitory action of maleimides reflects steric constraints imposed by the addition of a substituted alkyl group to the side chain of Cys634 rather than direct participation of this amino acid residue in catalysis.

Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

The vacuolar H(+)-pyrophosphatase (V-PPase) of plant cells is subject to substrate (Mg2PPi)-protectable, free PPi-potentiated irreversible inhibition by the sulfhydryl reagent N-ethylmaleimide (NEM). Inhibition by NEM approximates pseudo-first order kinetics and double-log plots of the first order rate constant for inactivation versus NEM concentration yield a straight line relationship with a slope of approximately unity. Since NEM and the membrane-impermeant cysteine reagent 3-(N-maleimidylpropionyl)biocytin (MPB) inhibit the V-PPase with similar kinetics and compete for a common binding site on the M(r) = 66,000 substrate-binding subunit, a single residue located in a cytosolically disposed extramembranous domain is inferred to undergo covalent modification in both cases. Selective labeling of the V-PPase of vacuolar membrane vesicles with [14C]NEM, purification of the M(r) = 66,000 subunit, and its digestion with V8 protease generates multiple peptide fragments. Of the bands identified after electrophoresis of the digests on Tris-Tricine gels, only one, migrating at M(r) = 14,000 (V814K), contains 14C label. Gas-phase sequence analysis of this band after electrotransfer to Immobilon PSQ yields two overlapping sequences (V814K2 and V814K2) which unambiguously align with the carboxyl-terminal segment of the M(r) = 66,000 subunit. Both V814K1 and V814K2 encompass only 1 cysteine residue at position 634 which is conserved between the V-PPases from Arabidopsis thaliana, Beta vulgaris (isoforms 1 and 2), and Hordeum vulgare. On the basis of these findings, the strict conservation of the sequence of the V-PPase from multiple plant sources, and the identical kinetics of interaction of the enzymes from Vigna and Beta with NEM and MPB, Cys634 of putative hydrophilic loop X is concluded to be the cytosolically oriented residue whose alkylation by maleimides is responsible for inactivation of the V-PPase. The significance of these results with respect to earlier speculations concerning the identity of the catalytic site and topology of the V-PPase is discussed.

Vacuolar membrane-derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H+-pyrophosphatase (V-PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme-bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V-PPase and soluble PPases suggests that the two classes of enzyme mediate PiHOH exchange by the same mechanism and that the intrinsic reversibility of the V-PPase is no greater than that of soluble PPases.

Phytochelatin synthase is the enzyme responsible for the synthesis of heavy-metal-binding peptides (phytochelatins) from glutathione and related thiols. It has recently been determined that it is not only restricted to plants and some fungi, as was once thought, but also has an essential role in heavy-metal detoxification in the model nematode Caenorhabditis elegans. These findings and others that demonstrate phytochelatin synthase-coding sequences in the genomes of several other invertebrates, including pathogenic nematodes, schistosomes and roundworms, herald a new era in phytochelatin research, in which these novel post-translationally synthesized peptides will not only be investigated in the context of phytoremediation but also from a clinical parasitological standpoint.

Supraoptimal levels of essential heavy metals (e.g., Cu and Zn) and trace or higher levels of nonessential heavy metals (e.g., As, Cd, and Hg) pose a threat because they undergo aberrant capping reactions with the thiol groups of proteins, displace endogenous metal cofactors from their cellular binding sites, and promote the formation of active oxygen species (1). One of the most sophisticated mechanisms known to confer protection from metal toxicity is the enzyme-catalyzed synthesis of short-chain peptides, phytochelatins (PCs), which serve as high-affinity, thiol-rich cellular chelators and thereby contribute to the detoxification of heavy metal ions, most notably Cd2+, in vascular plants and some algae, diatoms, fungi, and invertebrates (2). Derived from glutathione (GSH) and related thiols in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15) (1, 3, 4), PCs have the general structure (γ-Glu-Cys)n-Xaa, where n = 2–11 and Xaa is usually Gly (Fig. 1). Although studies of PC synthases have largely been concerned with the enzymes from eukaryotes, recent database searches have disclosed PC synthase-like sequences in the genomes of several prokaryotes (1, 5). In and of itself, this finding might not be of particular interest except that all of the prokaryotic PC synthase homologs identified are half the length of their cognates from eukaryotes (220–237 residues compared with 421–506 residues) because they lack the more sequence-variable C-terminal domain characteristic of the latter. Moreover, the one prokaryotic PC synthase homolog to have been assayed for activity, the alr0975 protein from the cyanobacterium Nostoc sp. PCC 7120 (NsPCS), catalyzes the deglycylation of GSH to γ-Glu-Cys at a high rate and the synthesis of PC2 at a relatively low rate (Fig. 1) (5, 6). It is as if the prokaryotic enzymes, despite an overall sequence identity of only 22–36% with the N-terminal domains of the eukaryotic enzymes, consist solely of the core catalytic domain of a canonical eukaryotic PC synthase (Fig. 1) (1, 7, 8). Against this background, the stereo structure of NsPCS in its native and γ-Glu-Cys-acylated state reported by Vivares et al. in a recent issue of PNAS (9) is seminal. The results presented not only represent the first crystal structure for a PC synthase but they also establish, as had been inferred tentatively from kinetic, protein chemical, and site mutagenic analyses of the prototypical eukaryotic PC synthase, Arabidopsis thaliana PC synthase 1 (AtPCS1) (Fig. 1) (1, 8, 10), that these enzymes belong to the papain superfamily and deploy a cysteine protease-like catalytic mechanism.

Publisher Summary This chapter discusses the molecular and biochemical basis of pyrophosphate–energized proton translocation at the vacuolar membrane. Inorganic pyrophosphatases (PPase) are ubiquitous enzymes that catalyze the hydrolysis of pyrophosphate to orthophosphate. In most organisms, the bulk of PPase activity is soluble and considered to maximize the energy yield from the pyrophosphorylytic cleavage of nucleoside triphosphates, associated with many biosynthetic reactions, by dissipatively hydrolysing PP i . Several lines of inquiry implicate PP i as an important energy source for plant vacuolar function in particular, and plant metabolism in general. Most of the H + phosphohydrolases associated with plant membranes can be distinguished from each other by their inhibitor sensitivities. The F–type H + –ATPases of energy–coupling membranes, the P–type H + –ATPase of plasma membrane and the V–type H + –ATPase of endomembranes are selectively inhibited by azide, orthovanadate and the bafilomycins, respectively. However, strict criteria for the identification of V–PPase activity in uncharacterized membrane fractions, other than through the measurement of azide–, orthovanadate– and bafilomycin–insensitive, K + –stimulated PP i –dependent H + translocation have been lacking. A major complication when attempting to determine the ligand interactions of any PPase, whether soluble or membrane bound, are the multiple ion complexes that PP, is capable of forming in aqueous media.

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.

The results of analyses of the steady-state kinetics of the vacuolar H(+)-translocating pyrophosphatase (V-PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata (mung bean) and purified enzyme from the same source under a wide range of Mg2+, pyrophosphate (PPi) and K+ concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme-Mg2+ complex. When account is taken of the sensitivity of the V-PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg2+ binding in the absence of PPi is an order of magnitude smaller than that estimated from the steady-state kinetics of PPi hydrolysis. Two distinct Mg(2+)-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cations and Mg2+ and K+ do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pKa 5.7 and 8.6 in substrate binding and groups of pKa 6.1 and 9.0 in the substrate-conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V-PPase and the potential regulatory significance of cytosolic free Mg2+ and K+ in vivo.

General acceptance of the chemiosmotic hypothesis (Mitchell 1961) has given rise to the view that membrane-bound H+-pumps constitute the primary transducers by means of which living cells inter-convert light, chemical and electrical energy. Through the establishment and maintenance of trans-membrane electrochemical gradients, H+ pumps energise the transport of other solutes or, in the special case of the energy-coupling membranes of mitochondria, chloroplasts and bacteria, transduce the H+-electrochemical gradient generated by membrane-linked anisotropic redox reactions to the synthesis of ATP (Harold, 1986). Given the multitude of biological reactions energised by ATP, primary H+-translocation and the inter-conversions of ATP have come to be recognised as the principal generators of usable energy in the cell. Against this background it is, therefore, surprising to find that the vacuolar membrane (tonoplast) of plant cells contains not only a V-(“vacuolar”) type H+ -ATPase (EC 3.6.1.3) (Rea and Sanders, 1987; Nelson and Taiz, 1989) but also an inorganic pyrophosphate- (PPi) energised H+ pump (V-Type H+-PPase; EC 3.6.1.1) (Rea and Sanders, 1987). Both enzymes catalyse inward electrogenic H+-PPase has the unusual characteristic of exclusively utilising PPi as energy source (Rea and Poole, 1986).

The vacuolar membrane (tonoplast) of plant cells contains two functionally and physically distinct phosphohydrolases, which catalyse electrogenic H+ -translocation: An ATPase (tp-ATPase; EC 3.6.1.3) and an inorganic pyrophosphatase (tp-PPase; 3.6.1.1). Neither enzyme belongs to the F0F1– or E1E2-categories of primary cation pumps, but instead belong to a third and fourth category of enzyme, respectively. Research priorities for the tp-ATPase are studies directed at understanding the roles of the 70 and 60 kDa subunits in catalysis and regulation; the involvement of the 16 kDa subunit in transmembrane H+ conduction; and investigations of F0F1- like structure/function partitioning. In the longer term, comparisons of sequence homology between the N,N′- dicyclohexylcarbodiimide -binding (16 kDa) proteins from different sources may enable elucidation of the evolutionary relationship of the tp-ATPase with other putative third-category H+– translocases. The tp-PPase, on the other hand, represents an exciting but largely unexplored biochemical entity, which necessitates a reconsideration of accepted views concerning the involvement of inorganic pyrophosphate (PPi) in transmembrane energy conservation. Just why the tonoplast should be endowed with two H+-translocases is a problem that can only be approached once consideration is given to the paramount question of H+/PPi stoichiometry. Once the stoichiometry is known, it should be possible to establish the physiological poise of the tp-PPase, and hence to speculate on its role in the metabolism of plant cells.

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20–25 µmol/mg•min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577–3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatographypurified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6–7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPases from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.

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.

The H+-ATPase of Beta vacuolar membrane (tonoplast) comprises at least three functionally distinct subunits of Mr = 67,000, 57,000, and 16,000, respectively (Manolson, M. F., Rea, P. A., and Poole, R. J. (1985) J. Biol. Chem. 260, 12273-12279). The hydrophobic carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the enzyme with pseudo-first order kinetics, and the concentration dependence of the reaction indicates that DCCD interacts with a single site on the enzyme to exert its inhibitory effect. The apparent pseudo-first order rate constant (k0) is reciprocally dependent on membrane protein concentration, which is expected if a large fraction of the DCCD partitions into the lipid phase. k0 has a nominal value of 1000 M-1 min-1 at a protein concentration of 250 micrograms/ml, although when phase partitioning is taken into account, the true, protein concentration-independent value of k0 is calculated to be about an order of magnitude lower. [14C]DCCD primarily labels the Mr = 16,000 polypeptide of native tonoplast vesicles. Binding is venturicidin-insensitive and occurs at a rate similar to the rate of enzyme inactivation, implying that inhibition is a direct result of covalent modification of the Mr = 16,000 polypeptide. Labeling of the containing Mr = 8,000 subunit of mitochondrial F0F1-ATPase is, on the other hand, faster by a factor of 5 and totally abolished by venturicidin. These results confirm that the Mr = 16,000 polypeptide which copurifies with tonoplast H+-ATPase activity is a subunit of the enzyme. Most of the DCCD-reactive Mr = 16,000 subunit is extracted from acetone:ethanol-washed tonoplast vesicles by chloroform:methanol. [14C]DCCD bound to the Mr = 16,000 polypeptide is enriched in the chloroform:methanol extract by 5-fold compared with native tonoplast and the specific activity (nmol of [14C]DCCD/mg of protein) can be increased a further 37-fold by chromatography on DEAE-Sephadex. It is concluded that the Mr = 16,000 subunit of the tonoplast H+-ATPase is a proteolipid.

The net uptake of 14C-D-alanine by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's flytrap) is positively correlated with the rate of H+ efflux from the trap lobes. Effectors that stimulate H+ efflux (secretion elicitor, fusicoccin and auxin) enhance D-alanine uptake whereas effectors that inhibit H+ efflux (diethylstilbestrol) retard uptake. It is proposed that amino acid uptake by the digestive glands of the trap lobes proceeds via a mechanism which mediates the co-transport of H+ ions and amino acids. A model is presented in which a positively charged ternary complex of carrier plus amino acid plus H+ is transported down an inwardly directed H+ electrochemical potential difference generated by the secretion-elicitor-provoked discharge of HCl into the trap cavity.

Membrane vesicles derived from the tonoplast of Beta vulgaris L. possess two predominant phosphohydrolase activities: a Cl − -stimulated, NO 3 − -inhibited ATPase, and a K + -stimulated, Na + -inhibited inorganic pyrophosphatase (PPase). The solubilization of tonoplast vesicles with 2% (w/v) Triton X-100 containing 4 millimolar MgCl 2 and 1 millimolar ethylenediamine tetracetic acid, as protectants, gives high yields of both the ATPase and PPase in soluble form. Chromatography of the solubilized membranes on Sephacryl-400 results in the separation of the two enzymes. The PPase and ATPase are purified 4- and 17-fold, respectively, with quantitative recovery. The separated enzymes show negligible activity towards the other9s substrate and the separated PPase only hydrolyzes pyrophosphate. The separated enzymes show mineral ion requirements identical to those of the corresponding pump and hydrolytic activities in native tonoplast and both solubilized enzymes are subject to phospholipid activation.

Summary The chemical requirements for the elicitation of the secretion of digestive fluid by the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap) were examined and the mineral ion and organic solute composition of the secreted fluid determined. A wide range of low molecular weight nitrogen compounds were found to cause the secretion of both Cl- and acid proteolytic activity when given at concentrations of 1 or 10 mM. Salts of the alkali metals were similarly effective while alkaline earth and transition metal salts and salt-free protein suspensions were inactive. The digestive fluid has an average osmolarity of 10–14 mosM. Cl- is usually present at 5–10 mEq and constitutes more than 90 per cent of the contribution of anions to the total osmolarity and between 70 and 100 per cent of the contribution of free anions to the electrical charge balance of the secretion. H+'s ore the principal monovalent cation in the fluid and are found at an average activity of 1–2 mEq while K+, Na+, Mg++ and Ca++ are each present at concentrations of less than 1 mM. Short-chain carboxylic acids, including formic acid, could not be detected in the secretion but low concentrations of glycerol, inositol, glucose, an unidentified dimeric sugar and several long-chain fatty acids were found. It is proposed that HC1 is responsible for the acidity of the digestive fluid and that a major effect of secretion elicitors is to initiate the discharge of this mineral acid from the digestive glands. The source and significance of the organic constituents of the secretion remains to be determined.

Simple kinetic techniques were used to examine the mechanism of D-alanine uptake by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap.) On the basis of these analyses, the uptake of D-alanine was found to depend on the time during which the trap lobes were inoculated with elicitors of secretion before excision and measurement of uptake. Disks taken from traps that had not been subjected to a preceding period of inoculation with secretion elicitors showed a low basal rate of uptake which was neither pH-dependent nor exhibited saturation with respect to external D-alanine concentration. Disks from preinoculated traps, on the other hand, displayed an enhanced rate of uptake which showed both pH-dependence and saturation with respect to external D-alanine concentration. The capacity for enhanced uptake was lost upon prolonged inoculation or when inoculation was stopped. Of the compounds tested, only elicitors of secretion caused an enhancement of uptake. The enhanced rate of D-alanine uptake is temperature-sensitive with a Q10 characteristic of a mediated process. Uncouplers cause an instantaneous abolition of uptake whereas the effects of terminal-oxidase inhibitors are time-dependent. The pH-dependence of uptake is inferred to result from an increased affinity of the carrier system for D-alanine at low pH values. Although the ionic state of D-alanine is relatively unaffected over the pH range examined, a decrease in the external pH from 6.0 to 3.8 decreases the apparent K m for uptake by four-fold but increases V max by only 30%. It is concluded that the acid secreted by the digestive glands of Dionaea plays a direct role in facilitating the uptake of amino acids from the trap cavity.

The H+-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl− and NO3−. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the Km of the enzyme for ATP but caused relatively little alteration of the Vmax. Nitrate increased Km only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl−. Other monovalent anions that specifically increased the Km of the ATPase for ATP were, in order of increasing Ki, SCN−, ClO4−, and ClO3−. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO3−, was a competitive inhibitor of the ATPase but ADP and NO3− did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO3− does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO3− exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl− nor NO3− are involved in substrate binding.