Search

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.

Does a microbiologist working on fungi who discovers a new class of drugs that had a global impact sound familiar? Would a Scottish researcher by the name of Alexander Fleming and the discovery of the penicillins and their development in the 1930s and 1940s be the first thing that comes to mind? Well, it should be but that is not the answer here. Instead, it is the Japanese researcher Akira Endo and the discovery of another class of fungal compounds, the statins, and their development in the 1970s and 1980s. Stated plainly, the discovery of the statins and the new insights into cardiovascular disease (CVD) that have come from their implementation represents one of the most significant accomplishments of the biomedical sciences in the twentieth century. A STARK REALITY CVD is the leading cause of death and disability in the developed world. As of 2010, the American Heart Association estimated that more than 80 million Americans have one or more forms of CVD. The stark reality is that more than 2,150 of us die of CVD each and every day and that once every 40 seconds someone in the Unites States has a terminal cardiovascular event. It may come as a surprise but CVD is the number one worldwide killer of women – a woman is 10 times more likely to die from heart disease than from breast cancer, the number one women's health news item (admittedly with good reason). The socioeconomic burden this represents is huge. More than 7.6 million CVD operations and procedures were performed in the United States in 2010, of which an estimated 3.6 million were performed on men and 2.6 million on women. The total direct and indirect costs for the American economy, inclusive of procedures, physicians and other health care professionals, prescribed medications and devices, home health care, and lost productivity in the same year is estimated at $315.4 billion. Daunting as these statistics are, the number of deaths in this part of the world has been on the decrease since the 1980s and is continuing to fall.

This volume began with the question of whether the process (e.g., pace and cost) of discovery in the biomedical sciences, and those individuals who make the discoveries, can or should be “managed” (see Chapter 1). To many, management suggests an organizational context that involves the provision of resources, incentives, and even bureaucratic controls. Indeed, the French root for manage is manege – to put a horse through its paces. Yet as Rosabeth Moss Kanter, former editor of the Harvard Business Review , observed long ago the innovation process is uncertain (e.g., unpredictable, nonlinear), tacit and fragile (requiring both individual creativity and interactive learning), controversial and political (e.g., competition among ideas), and boundary spanning (involving multiple disciplines, new connections, and exploration into new domains). This poses a seemingly paradoxical demand: while discovery and innovation are uncertain, tacit, political, and boundary-spanning processes they nevertheless somehow occur within a context of bureaucracy and managerial oversight. The paradox is easily resolved. Discoveries are made by individuals and groups, often (but not always) within and across organizations, and often supported and encouraged by those organizations – but not necessarily or typically controlled by those organizations. The micro-level processes and interactions that yield discoveries and innovations can be stimulated, facilitated, or enhanced by more macro-level departmental, company-wide, and inter-organizational actions and conditions – but only as long as these conditions take account of the entrepreneurial and disruptive nature of innovation. Such characteristics of innovation beg the question addressed in this volume of whether discovery can be managed. According to this micro/macro perspective, discovery takes place within a multi-tiered ecosystem. The ecosystem contains actors and dynamics at several levels of analysis: the individual inventor, teams or groups, the departments in which these individuals and teams are housed, the firms that bring these individuals and teams together, and the regions of the country where these firms locate and interact with local academic and governmental institutions (also known as inter-organizational “economic clusters”). At each level, there are multiple circumstances and forces at play that serve to spur (or hinder) discovery. Research on innovation in general, and discovery in the biomedical sciences in particular, has identified many of these forces.

In this book, distinguished scholars Philip A. Rea, Mark V. Pauly, and Lawton R. Burns explore the science and management behind marketable biomedical innovations. They look at how the science actually played out through the interplay of personalities, the cultures within and between academic and corporate entities, and the significance of serendipity not as a mysterious phenomenon but one intrinsic to the successes and failures of the experimental approach. With newly aggregated data and case studies, they consider the fundamental economic underpinnings of investor-driven discovery management, not as an obstacle or deficiency as its critics would contend or as something beyond reproach as some of its proponents might claim, but as the only means by which scientists and managers can navigate the unknowable to discover new products and decide how to sell them so as to maximize the likelihood of establishing a sustainable pipeline for still more marketable biomedical innovations.

Half-molecule ATP-binding cassette transporters of the HMT-1 (heavy metal tolerance factor 1) subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans, and Chlamydomonas reinhardtii. Based on studies of S. pombe, it has been proposed that SpHMT-1 transports heavy metal·phytochelatin (PC) complexes into the vacuolysosomal compartment. PCs are glutathione derivatives synthesized by PC synthases (PCS) in plants, fungi, and C. elegans in response to heavy metals. Our previous studies in C. elegans, however, suggested that HMT-1 and PCS-1 do not necessarily act in concert in metal detoxification. To further explore this inconsistency, we have gone on to test whether DmHMT-1, an HMT-1 from a new source, Drosophila, whose genome lacks PCS homologs, functions in heavy metal detoxification. In so doing, we show that heterologously expressed DmHMT-1 suppresses the Cd2+ hypersensitivity of S. pombe hmt-1 mutants and localizes to the vacuolar membrane but does not transport Cd·PC complexes. Crucially, similar analyses of S. pombe hmt-1 mutants extend this finding to show that SpHMT-1 itself either does not transport Cd·PC complexes or is not the principal Cd·PC/apoPC transporter. Consistent with this discovery and with our previous suggestion that HMT-1 and PCS-1 do not operate in a simple linear metal detoxification pathway, we demonstrate that, unlike PCS-deficient cells, which are hypersensitive to several heavy metals, SpHMT-1-deficient cells are hypersensitive to Cd2+, but not to Hg2+ or As3+. These findings significantly change our current understanding of the function of HMT-1 proteins and invoke a PC-independent role for these transporters in Cd2+ detoxification.

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of ‘proteomics-grade’ intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione–bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.

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.

This Update is concerned with the mechanism of synthesis of heavy metal-binding thiol peptides, phytochelatins (PCs), by the enzyme PC synthase (EC 2.3.2.15). The bulk of the considerations in this review centers on what has been learned recently of the fundamental mechanics of PC synthesis, the domain organization and phylogenetic distribution of PC synthases, and PC synthase-like enzymes, and what this tells us about the chemistry underlying and the enzyme residues necessary for PC synthesis. It was decided to prepare a review of this type rather than aim at a more comprehensive treatment of heavy metal homeostasis and detoxification in plants for two reasons. The first is that there are already several contemporary reviews dealing with the more global aspects of plant heavy metal physiology. Excellent examples are Cobbett (2000), Clemens (2001), and Cobbett and Goldsbrough (2002). Readers who have not already read these are encouraged to do so. The second reason is that some of the most fascinating and unexpected developments for our understanding in this area of late derive from investigations of the catalytic mechanism and distribution of PC synthases, facets of this field of research that have yet to be reviewed in detail.

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.

Plant nutrition critically depends on the activity of membrane transporters that translocate minerals from the soil into the plant and are responsible for their intra- and intercellular distribution. Most plant membrane transporters are encoded by multigene families whose members often exhibit overlapping expression patterns and a high degree of sequence homology. Furthermore, many inorganic nutrients are transported by more than one transporter family. These considerations, coupled with a large number of so-far non-annotated putative transporter genes, hamper our progress in understanding how the activity of specific transporters is integrated into a response to fluctuating conditions. We designed an oligonucleotide microarray representing 1096 Arabidopsis transporter genes and analysed the root transporter transcriptome over a 96-h period with respect to 80 mM NaCl, K+ starvation and Ca2+ starvation. Our data show that cation stress led to changes in transcript level of many genes across most transporter gene families. Analysis of transcriptionally modulated genes across all functional groups of transporters revealed families such as V-type ATPases and aquaporins that responded to all treatments, and families - which included putative non-selective cation channels for the NaCl treatment and metal transporters for Ca2+ starvation conditions - that responded to specific ionic environments. Several gene families including primary pumps, antiporters and aquaporins were analysed in detail with respect to the mRNA levels of different isoforms during ion stress. Cluster analysis allowed identification of distinct expression profiles, and several novel putative regulatory motifs were discovered within sets of co-expressed genes.

Here we report the isolation and characterization of a type I vacuolar-type H+-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan,Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K+-activated pyrophosphate (PPi) hydrolysis and PPi-energized H+ translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 μm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I50 for the inhibition of TgVP1 activity in vitro (0.9 μm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.

Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H+gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H+ gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H+ gradient dependence of glucoside uptake. It is concluded that H+gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H+ antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.

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.

Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, γ-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that thece-pcs-1 gene of the nematode wormCaenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.

In plants, cytosolic inorganic pyrophosphate (PP(i)) is hydrolyzed by energy-conserving vacuolar-type H(+)-pyrophosphatases (V-PPases) that harness the free energy of PP(i) hydrolysis to establish transmembrane H(+) gradients. Here we describe the identification and cloning of two genes, PfVP1 and PfVP2, from the malaria parasite Plasmodium falciparum. Inferred to encode type I (K(+)-dependent) and type II (K(+)-independent) V-PPases, respectively, PfVP1 and PfVP2 appeared more sequence divergent from each other than from their type I and type II counterparts in plants. The steady state levels of PfVP1 mRNA were high in comparison to PfVP2 mRNA throughout the erythrocytic phases of infection. Western analyses of trophozoite membranes using generic V-PPase antibodies (PAB(HK) and PAB(TK)) demonstrated appreciable amounts of a Mr 67000 polypeptide whose associated aminomethylenediphosphonate- (AMDP) inhibitable PPase activity was markedly stimulated by K(+). Immunofluorescence microscopy of infected erythrocytes revealed PfVP antigen associated with both the parasite plasma membrane and punctate intracellular inclusions. Transient transfection of a PfVP1-GFP fusion further supported the localization of PfVP1 to the parasite plasma membrane. Based on these findings and the growth-retarding effects of AMDP, P. falciparum is concluded to possess both type I and type II V-PPases of which the former has the greatest potential for contributing to the establishment of H(+) gradients across the parasite plasma membrane under conditions of energy limitation.

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E(2)17betaG uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E(2)17betaG uptake, neither competes with DNP-GS for uptake nor is subject to E(2)17betaG-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin-Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1-3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.

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.

This review addresses recent advances in the understanding of the function and organization of one subfamily of ATP-binding cassette (ABC) transporters, the multidrug resistance-associated proteins (MRPs), from plants and yeast. So named because of the phenotypes conferred by their animal prototypes, many MRPs are glutathione-conjugate or multi-specific organic anion Mg 2+ -ATPases active in the transport of glutathionated compounds and other bulky amphipathic anions. Acting downstream of the first two phases of toxin detoxification, typified by, but not limited to the reactions catalysed by the cytochromes P450 and glutathione S-transferases, respectively, processes that likely converge and depend on the MRPs from plants and yeast include the detoxification of herbicides and other organic xenobiotics, the alleviation of oxidative damage, the storage of endotoxins, heavy metal sequestration, and the vacuolation of natural pigments. Distinguished by a unique domain organization and the facility to mediate MgATP-energized transport, the MRPs are of profound significance in two respects. First, the fact that the MRPs, like most ABC transporters, are energized directly by MgATP rather than by, for example, a preformed H + gradient, offers a new, non-chemiosmotic perspective on the molecular basis of energy-dependent solute transport in plants. Second, given the existence of 29 ABC protein genes in the yeast genome alone, characterization of the MRPs-the first class of ABC transporters from a plant source to have been defined at both the molecular and biochemical levels-indicates that many more ABC transporters and ABC transporter-mediated processes remain to be discovered in plants.

▪ Abstract While the concept of H+-coupling has dominated studies of energy-dependent organic solute transport in plants for over two decades, recent studies have demonstrated the existence of a group of organic solute transporters, belonging to the ATP-binding cassette (ABC) superfamily, that are directly energized by MgATP rather than by a transmembrane H+-electrochemical potential difference. Originally identified in microbial and animal cells, the ABC superfamily is one of the largest and most widespread protein families known. Competent in the transport of a broad range of substances including sugars, peptides, alkaloids, inorganic anions, and lipids, all ABC transporters are constituted of one or two copies each of an integral membrane sector and cytosolically oriented ATP-binding domain. To date, two major subclasses, the multidrug resistance-associated proteins (MRPs) and multidrug resistance proteins (MDRs) (so named because of the phenotypes conferred by their animal prototypes), have been identified molecularly in plants. However, only the MRPs have been defined functionally. This review therefore focuses on the functional capabilities, energetics, organization, and regulation of the plant MRPs. Otherwise known as GS-X pumps, or glutathione-conjugate or multispecific organic anion Mg2+-ATPases, the MRPs are considered to participate in the transport of exogenous and endogenous amphipathic anions and glutathionated compounds from the cytosol into the vacuole. Encoded by a multigene family and possessing a unique domain organization, the types of processes that likely converge and depend on plant MRPs include herbicide detoxification, cell pigmentation, the alleviation of oxidative damage, and the storage of antimicrobial compounds. Additional functional capabilities might include channel regulation or activity, and/or the transport of heavy metal chelates. The identification of the MRPs, in particular, and the demonstration of a central role for ABC transporters, in general, in plant function not only provide fresh insights into the molecular basis of energy-dependent solute transport but also offer the prospect for manipulating and investigating many fundamental processes that have hitherto evaded analysis at the transport level.

Three ATP binding cassette (ABC) transporter-like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP-energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins.

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.

Because plants produce cytotoxic compounds to which they, themselves, are susceptible and are exposed to exogenous toxins (microbial products, allelochemicals, and agrochemicals), cell survival is contingent on mechanisms for detoxifying these agents. One detoxification mechanism is the glutathione S -transferase-catalyzed glutathionation of the toxin, or an activated derivative, and transport of the conjugate out of the cytosol. We show here that a transporter responsible for the removal of glutathione S -conjugates from the cytosol, a specific Mg 2+ -ATPase, is encoded by the AtMRP1 gene of Arabidopsis thaliana . The sequence of AtMRP1 and the transport capabilities of membranes prepared from yeast cells transformed with plasmid-borne AtMRP1 demonstrate that this gene encodes an ATP-binding cassette transporter competent in the transport of glutathione S -conjugates of xenobiotics and endogenous substances, including herbicides and anthocyanins.

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.

This review addresses the recent molecular identification of several members of the glutathione S-conjugate (GS-X) pump family, a new class of ATP-binding cassette (ABC) transporters responsible for the elimination and/or sequestration of pharmacologically and agronomically important compounds in mammalian, yeast and plant cells. The molecular structure and function of GS-X pumps encoded by MRP, cMOAT, YCF1. and AtMRP genes, have been conserved throughout molecular evolution. The physiologic function of GS-X pumps is closely related with cellular detoxification, oxidative stress, inflammation, and cancer drug resistance. Coordinated expression of GS-X pump genes, e.g., MRP1 and YCF1, and γ-glutamylcystaine synthetase, a rate-limiting enzyme of cellular glutathione (GSH) biosynthesis, has been frequently observed.

The yeast cadmium factor ( YCF1 ) gene encodes an MgATP-energized glutathione S -conjugate transporter responsible for the vacuolar sequestration of organic compounds after their S -conjugation with glutathione. However, while YCF1 was originally isolated according to its ability to confer resistance to cadmium salts, neither its mode of interaction with Cd 2+ nor the relationship between this process and organic glutathione-conjugate transport are known. Here we show through direct comparisons between vacuolar membrane vesicles purified from Saccharomyces cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild-type strain DTY165 that YCF1 mediates the MgATP-energized vacuolar accumulation of Cd·glutathione complexes. The substrate requirements, kinetics and Cd 2+ /glutathione stoichiometry of cadmium uptake and the molecular weight of the transport-active complex demonstrate that YCF1 selectively catalyzes the transport of bis(glutathionato)cadmium (Cd·GS 2 ). On the basis of these results—the Cd 2+ hypersensitivity of DTY167, versus DTY165, cells, the inducibility of YCF1-mediated transport, and the rapidity and spontaneity of Cd·GS 2 formation—this new pathway is concluded to contribute substantially to Cd 2+ detoxification.

A plant gene that confers antibiotic resistance provides a 'cleaner' selectable marker for plant transgenesis.

By characterization of the uptake of glutathione-S-conjugates, principally dinitrophenyl-S-glutathione (DNP-GS), by vacuolar membrane vesicles, we demonstrate that a subset of energy-dependent transport processes in plants are not H+-coupled but instead are directly energized by MgATP. The most salient features of this transport pathway are: (a) its specific, obligate requirement for MgATP as energy source; (b) the necessity for hydrolysis of the [gamma]-phosphate of MgATP for uptake; (c) the insensitivity of uptake to uncouplers of the transtonoplast H+ gradient (carbonylcyanide 4-trifluoromethoxyphenylhydrazone, gramicidin-D, and NH4Cl); (d) its pronounced sensitivity to vanadate and partial inhibition by vinblastine and verapamil; (e) the lack of chemical modification of DNP-GS either during or after transport; (f) the capacity of S-conjugates of chloroacetanilide herbicides, such as metolachlor-GS, but not free herbicide, to inhibit uptake; and (g) the ability of vacuolar membrane vesicles purified from a broad range of plant species, including Arabidopsis, Beta, Vigna, and Zea, to mediate MgATP-dependent, H+-electrochemical potential difference-independent DNP-GS uptake. On the basis of these findings it is proposed that the transport of DNP-GS across the vacuolar membrane of plant cells is catalyzed by a glutathione-conjugate transporter that directly employs MgATP rather than the energy contained in the transtonoplast H+-electrochemical potential difference to drive uptake. The broad distribution of the vacuolar DNP-GS transporter and its inhibition by metolachlor-GS are consistent with the notion that it plays a general role in the vacuolar sequestration of glutathione-conjugable cytotoxic agents.

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.