Uptake of vanadium and its intracellular metabolism by Coprinellus truncorum mycelial biomass.

BACKGROUND: Fungi absorb and solubilize a broad spectrum of heavy metals such as vanadium (V), which makes them a main route of its entry into the biosphere. V as vanadate (V5+) is a potential medical agent due to its many metabolic actions such as interaction with phosphates in the cell, and especially its insulin-mimetic activity. Antidiabetic activity of V-enriched fungi has been studied in recent years, but the biological and chemical bases of vanadium action and status in fungi in general are poorly understood, with almost no information on edible fungi. METHODS: This manuscript gives a deeper insight into the interaction of V5+ with Coprinellus truncorum, an edible autochthonous species widely distributed in Europe and North America. Vanadium uptake and accumulation as V5+ was studied by 51V NMR, while the reducing abilities of the mycelium were determined by EPR. 31P NMR was used to determine its effects on the metabolism of phosphate compounds, with particular focus on phosphate sugars identified using HPLC. RESULTS: Vanadate enters the mycelium in monomeric form and shows no immediate detrimental effects on intracellular pH or polyphosphate (PPc) levels, even when applied at physiologically high concentrations (20 mM Na3VO4). Once absorbed, it is partially reduced to less toxic vanadyl (V4+) with notable unreduced portion, which leads to a large increase in phosphorylated sugar levels, especially glucose-1-phosphate (G1P) and fructose-6-phosphate (F6P). CONCLUSIONS: Preservation of pH and especially PPc reflects maintenance of the energy status of the mycelium, i.e., its tolerance to high V5+ concentrations. Rise in G1P and F6P levels implies that the main targets of V5+ are most likely phosphoglucomutase and phosphoglucokinase(s), enzymes involved in early stages of G6P transformation in glycolysis and glycogen metabolism. This study recommends C. truncorum for further investigation as a potential antidiabetic agent.

An innovative in situ AFM system for a soft X-ray spectromicroscopy synchrotron beamline.

Multimodal imaging and spectroscopy like concurrent scanning transmission X-ray microscopy (STXM) and X-ray fluorescence (XRF) are highly desirable as they allow retrieving complementary information. This paper reports on the design, development, integration and field testing of a novel in situ atomic force microscopy (AFM) instrument for operation under high vacuum in a synchrotron soft X-ray microscopy STXM-XRF end-station. A combination of µXRF and AFM is demonstrated for the first time in the soft X-ray regime, with an outlook for the full XRF-STXM-AFM combination.

Investigation of novel radical scavenging mechanisms in the alkaline environment: Green, sustainable and environmentally friendly antioxidative agent(s).

Pharmaceutical and industrial utilization of synthetic chemicals has an immerse impact on the environment. In that sense, novel chemicals with potential for industrial application should be investigated for their behaviour in reactions with hydroxyl radical, simulating AOPs (Advanced Oxidation Processes). AOPs are known for being highly effective in wastewater management and natural water remediation. In this paper, exhaustive research on the radical scavenging activity of a newly synthesized coumarin derivative (4HCBH), as a representative of the series of coumarin-benzohydrazides with high antioxidative potential was conducted. This study took into consideration the pH value range significant for practically all living organisms (pH = 7.0-8.5). According to the experimentally obtained results, the 4HCBH showed an increase in radical scavenging activity, following the slight increase in pH values, which suggested that the formation of anionic form of 4HCBH is responsible for its antiradical activity. Further investigations led to the postulation of a novel mechanistic approach called Sequential Proton Loss Electron Transfer - Radical-Radical Coupling (SPLET-RRC), in which, by a series of steps, a new, stable compound was formed. Furthermore, it was demonstrated that the product generated through SPLET-RRC showed lower toxicity than the parent molecule.

Degradation Mechanisms of 4,7-Dihydroxycoumarin Derivatives in Advanced Oxidation Processes: Experimental and Kinetic DFT Study.

Coumarins represent a broad class of compounds with pronounced pharmacological properties and therapeutic potential. The pursuit of the commercialization of these compounds requires the establishment of controlled and highly efficient degradation processes, such as advanced oxidation processes (AOPs). Application of this methodology necessitates a comprehensive understanding of the degradation mechanisms of these compounds. For this reason, possible reaction routes between HO• and recently synthesized aminophenol 4,7-dihydroxycoumarin derivatives, as model systems, were examined using electron paramagnetic resonance (EPR) spectroscopy and a quantum mechanical approach (a QM-ORSA methodology) based on density functional theory (DFT). The EPR results indicated that all compounds had significantly reduced amounts of HO• radicals present in the reaction system under physiological conditions. The kinetic DFT study showed that all investigated compounds reacted with HO• via HAT/PCET and SPLET mechanisms. The estimated overall rate constants (koverall) correlated with the EPR results satisfactorily. Unlike HO• radicals, the newly formed radicals did not show (or showed negligible) activity towards biomolecule models representing biological targets. Inactivation of the formed radical species through the synergistic action of O2/NOx or the subsequent reaction with HO• was thermodynamically favored. The ecotoxicity assessment of the starting compounds and oxidation products, formed in multistage reactions with O2/NOx and HO•, indicated that the formed products showed lower acute and chronic toxicity effects on aquatic organisms than the starting compounds, which is a prerequisite for the application of AOPs procedures in the degradation of compounds.

A three-step process of manganese acquisition and storage in the microalga Chlorella sorokiniana.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese-calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Biotransformation of selenium in the mycelium of the fungus Phycomyces blakesleeanus.

Biotransformation of toxic selenium ions to non-toxic species has been mainly focused on biofortification of microorganisms and production of selenium nanoparticles (SeNPs), while far less attention is paid to the mechanisms of transformation. In this study, we applied a combination of analytical techniques with the aim of characterizing the SeNPs themselves as well as monitoring the course of selenium transformation in the mycelium of the fungus Phycomyces blakesleeanus. Red coloration and pungent odor that appeared after only a few hours of incubation with 10 mM Se+4 indicate the formation of SeNPs and volatile methylated selenium compounds. SEM-EDS confirmed pure selenium NPs with an average diameter of 57 nm, which indicates potentially very good medical, optical, and photoelectric characteristics. XANES of mycelium revealed concentration-dependent mechanisms of reduction, where 0.5 mM Se+4 led to the predominant formation of Se-S-containing organic molecules, while 10 mM Se+4 induced production of biomethylated selenide (Se-2) in the form of volatile dimethylselenide (DMSe) and selenium nanoparticles (SeNPs), with the SeNPs/DMSe ratio rising with incubation time. Several structural forms of elemental selenium, predominantly monoclinic Se8 chains, together with trigonal Se polymer chain, Se8 and Se6 ring structures, were detected by Raman spectroscopy.

Mechanism of Antiradical Activity of Newly Synthesized 4,7-Dihydroxycoumarin Derivatives-Experimental and Kinetic DFT Study.

Coumarin derivatives have proven beneficial biological activities, but the mechanism of their radical scavenging potency is not fully understood. In this study, the antiradical capacity of two newly synthesized 4,7-dihydroxycoumarin derivatives: (E)-3-(1-((3-hydroxy-4-methoxyphenyl)amino)-ethylidene)-2,4-dioxochroman-7-yl acetate (A-3OH) and (E)-3-(1-((4-hydroxy-3-methoxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (A-4OH) towards HO• were examined by Electron Paramagnetic Resonance (EPR) Spectroscopy and Density Functional Theory (DFT). The compounds were fully characterized by the elemental microanalysis, IR, and NMR spectroscopies. The effect of pH on the acid-base equilibria is separately discussed and the predominant species at the physiological pH were determined. Several common mechanisms (Hydrogen Atom Transfer (HAT), Single-Electron Transfer followed by Proton Transfer (SET-PT), Sequential Proton Loss followed by Electron Transfer (SPLET), Radical Adduct Formation (RAF), and Intramolecular Hydrogen Atom Abstraction (iHAA)) of radical scavenging were investigated based on thermodynamic and kinetic parameters. EPR results indicated that both compounds significantly reduce the amount of present HO•. The results of the kinetic DFT study demonstrated that both compounds predominantly exhibit antiradical capacity through HAT and SPLET mechanisms. The estimated overall rate constants (koverall) proved that A-4OH shows better antioxidant capacity than A-3OH which is well-correlated with the results obtained by EPR measurement.

Synthesis and Biological Screening of New 4-Hydroxycoumarin Derivatives and Their Palladium(II) Complexes.

Two newly synthesized 4-hydroxycoumarin bidentate ligands (L1 and L2) and their palladium(II) complexes (C1 and C2) were screened for their biological activities, in vitro and in vivo. Structures of new compounds were established based on elemental analysis, 1H NMR, 13C NMR, and IR spectroscopic techniques. The obtained compounds were tested for their antioxidative and cytotoxic activities and results pointed to selective antiradical activity of palladium(II) complexes towards •OH and -•OOH radicals and anti-ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical) activity comparable to that of ascorbate. Results indicated the effect of C1 and C2 on the enzymatic activity of the antioxidative defense system. In vitro cytotoxicity assay performed on different carcinoma cell lines (HCT166, A375, and MIA PaCa-2), and one healthy fibroblast cell line (MRC-5) showed a cytotoxic effect of both C1 and C2, expressed as a decrease in carcinoma cells' viability, mostly by induction of apoptosis. In vivo toxicity tests performed on zebrafish embryos indicated different effects of C1 and C2, ranging from adverse developmental effect to no toxicity, depending on tested concentration. According to docking studies, both complexes (C1 and C2) showed better inhibitory activity in comparison to other palladium(II) complexes.

Mechanisms of detoxification of high copper concentrations by the microalga Chlorella sorokiniana.

Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper.

Biliverdin-copper complex at physiological pH.

Biliverdin (BV), a product of heme catabolism, is known to interact with transition metals, but the details of such interactions under physiological conditions are scarce. Herein, we examined coordinate/redox interactions of BV with Cu2+ in phosphate buffer at pH 7.4, using spectrophotometry, HESI-MS, Raman spectroscopy, 1H NMR, EPR, fluorimetry, and electrochemical methods. BV formed a stable coordination complex with copper in 1 : 1 stoichiometry. The structure of BV was more planar and energetically stable in the complex. The complex showed strong paramagnetic effects that were attributed to an unpaired delocalized e-. The delocalized electron may come from BV or Cu2+, so the complex is formally composed either of BV radical cation and Cu1+ or of BV radical anion and Cu3+. The complex underwent oxidation only in the presence of both O2 and an excess of Cu2+, or a strong oxidizing agent, and it was resistant to reducing agents. The biological effects of the stable BV metallocomplex containing a delocalized unpaired electron should be further examined, and may provide an answer to the long-standing question of high energy investment in the catabolism of BV, which represents a relatively harmless molecule per se.

Effects of vanadate on the mycelium of edible fungus Coprinus comatus.

Vanadate is proposed to play a pivotal role in application of edible fungus Coprinus comatus for medical purposes. In this study the concentration of extracellular vanadate acceptable for the submerged cultivation of C. comatus mycelium was established. The mycelium could grow, and overcome vanadate toxic effects, up to the concentration of 3.3 mM. Moreover, in this condition, at the end of the exponential phase of growth, biomass yield was almost identical to that in the control. 31P NMR spectroscopy showed that addition of 10 mM vanadate to the mycelium in the exponential phase of growth provoked instantaneous increase of a sugar phosphates level which could be related to changes in activities of glycolytic enzymes. Exposure to higher vanadate concentration was toxic for the cell. 51V NMR measurements revealed that monomer of vanadate is present in the cytoplasm causing the metabolic changes. C. comatus has also capacity for vanadate reduction, as shown by EPR measurements, but vanadyl uptake is significantly less comparing to vanadate.

Coordinate and redox interactions of epinephrine with ferric and ferrous iron at physiological pH.

Coordinate and redox interactions of epinephrine (Epi) with iron at physiological pH are essential for understanding two very different phenomena - the detrimental effects of chronic stress on the cardiovascular system and the cross-linking of catecholamine-rich biopolymers and frameworks. Here we show that Epi and Fe3+ form stable high-spin complexes in the 1:1 or 3:1 stoichiometry, depending on the Epi/Fe3+ concentration ratio (low or high). Oxygen atoms on the catechol ring represent the sites of coordinate bond formation within physiologically relevant bidentate 1:1 complex. Redox properties of Epi are slightly impacted by Fe3+. On the other hand, Epi and Fe2+ form a complex that acts as a strong reducing agent, which leads to the production of hydrogen peroxide via O2 reduction, and to a facilitated formation of the Epi-Fe3+ complexes. Epi is not oxidized in this process, i.e. Fe2+ is not an electron shuttle, but the electron donor. Epi-catalyzed oxidation of Fe2+ represents a plausible chemical basis of stress-related damage to heart cells. In addition, our results support the previous findings on the interactions of catecholamine moieties in polymers with iron and provide a novel strategy for improving the efficiency of cross-linking.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

The interactions of vanadate monomer with the mycelium of fungus Phycomyces blakesleeanus: reduction or uptake?

The possibility of reduction of vanadate monomer in the mycelium of fungus Phycomyces blakesleeanus was investigated in this study by means of polarography. Control experiments were performed with vanadyl [V(IV)] and vanadate [V(V)] in 10 mM Hepes, pH 7.2. Addition of P. blakesleeanus mycelium resulted in disappearance of all V(IV) polarographic waves recorded in the control. This points to the uptake of all available V(IV) by the mycelium, up to 185 µmol/gFW, and suggests P. blakesleeanus as a potential agent in V(IV) bioremediation. Polarographic measurements of mycelium with low concentrations (0.1-1 mM) of V(V), that only allows the presence of monomer, showed that fungal mycelia removes around 27% of V(V) from the extracellular solution. Uptake was saturated at 104 ± 2 µmol/gFW which indicates excellent bioaccumulation capability of P. blakesleeanus. EPR, 51V NMR and polarographic experiments showed no indications of any measurable extracellular complexation of V(V) monomer with fungal exudates, reduction by the mycelium or adsorption to the cell wall. Therefore, in contrast to vanadium oligomers, vanadate monomer interactions with the mycelium are restricted to its transport into the fungal cell, probably by a phosphate transporter.

(51)V NMR investigation of cell-associated vanadate species in Phycomyces blakesleeanus mycelium.

(51)V NMR spectroscopy was used for detection and identification of cell-associated vanadate (V(5+)) species after exposure of Phycomyces blakesleeanus mycelium, in exponential phase of growth, to sodium orthovanadate. Complete disappearance of monomer and dimer signals and decreased intensity of the tetramer signal were observed about 40 min after treatment. Simultaneously, a signal at -532 ppm, with increasing intensity, was detected in spectra. The time-dependent rise in this signal was connected to a decrease in the extracellular monomer signal, indicating its transport into the cell. The signal at -532 ppm did not belong to any known simple oxido-vanadate species, nor to a complex with any of the components of experimental medium. This signal was the only one present in spectrum of the mycelium washed 35 min after treatment, and the only one observed in mycelium cultivated on vanadate-contained medium. Therefore, its appearance can be attributed to intracellular complexation, and may represent an important detoxification mechanism of the cell exposed to a physiologically relevant concentration of vanadate. Experiments ((51)V NMR and polarography) performed with Cd-pretreated mycelium (inhibitor of an enzyme responsible for V(5+) reduction) and ferricyanide-preincubated mycelium excluded the possibility of V(5+) tetramer's entry into the cell.

X-ray absorption near-edge structure micro-spectroscopy study of vanadium speciation in Phycomyces blakesleeanus mycelium.

Vanadium speciation in the fungus Phycomyces blakesleeanus was examined by X-ray absorption near-edge structure (XANES) spectroscopy, enabling assessment of oxidation states and related molecular symmetries of this transition element in the fungus. The exposure of P. blakesleeanus to two physiologically important vanadium species (V(5+) and V(4+)) resulted in the accumulation of this metal in central compartments of 24 h old mycelia, most probably in vacuoles. Tetrahedral V(5+), octahedral V(4+), and proposed intracellular complexes of V(5+) were detected simultaneously after addition of a physiologically relevant concentration of V(5+) to the mycelium. A substantial fraction of the externally added V(4+) remained mostly in its original form. However, observable variations in the pre-edge-peak intensities in the XANES spectra indicated intracellular complexation and corresponding changes in the molecular coordination symmetry. Vanadate complexation was confirmed by (51)V NMR and Raman spectroscopy, and potential binding compounds including cell-wall constituents (chitosan and/or chitin), (poly)phosphates, DNA, and proteins are proposed. The evidenced vanadate complexation and reduction could also explain the resistance of P. blakesleeanus to high extracellular concentrations of vanadium.

Osmotic swelling activates a novel anionic current with VRAC-like properties in a cytoplasmic droplet membrane from Phycomyces blakesleeanus sporangiophores.

We describe here whole-cell currents of droplets prepared from the apical region of growing Phycomyces blakesleeanus sporangiophores. Whole-cell current recordings revealed the osmotically activated, outwardly rectifying, fast inactivating instantaneous current (ORIC) with biophysical properties closely resembling volume-regulated anionic current (VRAC). ORIC is activated under conditions of osmotically induced swelling and shows strong selectivity for anions over cations. In addition, ORIC shows voltage and time-dependent inactivation at positive potentials and recovery from inactivation at negative potentials. ORIC is blocked by anthracene-9-carboxylic acid, an anion channel blocker, in a voltage-dependent manner. This is the first report of the presence of VRAC-like current in an organism outside the chordate lineage.

Vanadate influence on metabolism of sugar phosphates in fungus Phycomyces blakesleeanus.

The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+), indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.

Oxygen regulation of alternative respiration in fungus Phycomyces blakesleeanus: connection with phosphate metabolism.

Environmental changes can often result in oxygen deficiency which influences cellular energy metabolism, but such effects have been insufficiently studied in fungi. The effects of oxygen deprivation on respiration and phosphate metabolites in Phycomyces blakesleeanus were investigated by oxygen electrode and (31)P NMR spectroscopy. Mycelium was incubated in hypoxic and anoxic conditions for 1.5, 3 and 5 h and then reoxygenated. Participation of alternative oxidase (AOX) in total respiration increased gradually in both treatments and after 5 h of anoxia exceeded a value 50% higher than in control. Shortly after reintroduction of oxygen into the system AOX level decreased close to the control level. Oxygen deprivation also caused a reversible decrease of polyphosphate/inorganic phosphate ratio (PPc/Pi), which was strongly correlated with the increase of AOX participation in total respiration. Unexpectedly, ATP content remained almost constant, probably due to the ability of PolyP to sustain energy and phosphate homeostasis of the cell under stress conditions. This was further substantiated by the effects of azide, a cytochrome c oxidase inhibitor, which also decreased PPc/Pi ratio, but to a smaller extent in oxygen deprived than control and reoxygenated specimens.

The interactions of vanadium with Phycomyces blakesleeanus mycelium: enzymatic reduction, transport and metabolic effects.

The biological and chemical basis of vanadium action and transport in fungi is relatively poorly understood. In this study we investigated the interactions of vanadium in physiologically-relevant redox states: vanadate (+5) and vanadyl (+4), with mycelium of fungus Phycomyces blakesleeanus using EPR and (31)P NMR spectroscopy and biochemical assays. We determined that P. blakesleeanus reduces V(5+) to V(4+) in the extracellular compartment by the means of cell surface enzyme with ferricyanide reductase activity, which contains molybdenum-molybdopterin as a cofactor. Both, V(5+) and V(4+) bind to cell wall. They enter the cytoplasm via phosphate transporter and cation channels, respectively, and exhibit different metabolic effects. Vanadate provokes increased biomass production, the effects being inverted to toxic at higher V(5+) concentrations. In addition, V(5+) activates the synthesis of sugar phosphates and oligophosphates. On the other hand, V(4+) exhibits toxic effects even at low concentrations. The V(4+) detoxification route involves binding to vacuolar polyphosphates. Altogether our results imply that the mechanism of interaction of vanadium with P. blakesleeanus involves three major steps: extracellular enzymatic V(5+)/V(4+) reduction, V(4+) influx, and vacuolar storage, with an additional step - V(5+) import occurring at higher vanadate concentrations.