Oxidation of arsenite to arsenate by Alcaligenes faecalis.

Alcaligenes faecalis, resistant to the toxic effects of 0.01 M sodium arsenite, was isolated from raw sewage and shown to be capable of oxidizing arsenite to arsenate. When the organisms were grown in chemically defined medium, this conversion was due to the appearance at stationary phase of an intracellular, oxygen-sensitive, inducible enzyme and/or component of the electron transport system; when the organisms were grown in a nutrient broth-yeast extract medium, the enzyme appeared in the late exponential phase of growth. The presence of 0.02 M arsenite in the culture medium affected neither growth rate nor final cell yield.     

Oxidation of arsenite to arsenate by Alcaligenes faecalis.

Alcaligenes faecalis, resistant to the toxic effects of 0.01 M sodium arsenite, was isolated from raw sewage and shown to be capable of oxidizing arsenite to arsenate. When the organisms were grown in chemically defined medium, this conversion was due to the appearance at stationary phase of an intracellular, oxygen-sensitive, inducible enzyme and/or component of the electron transport system; when the organisms were grown in a nutrient broth-yeast extract medium, the enzyme appeared in the late exponential phase of growth. The presence of 0.02 M arsenite in the culture medium affected neither growth rate nor final cell yield.     

Inhibition of microbial arsenate reduction by phosphate

The ratio of arsenite (As(III)) to arsenate (As(V)) in soils and natural waters is often controlled by the activity of As-transforming microorganisms. Phosphate is a chemical analog to As(V) and, consequently, may competitively inhibit microbial uptake and enzymatic binding of As(V), thus preventing its reduction to the more toxic, mobile, and bioavailable form - As(III). Five As-transforming bacteria isolated either from As-treated soil columns or from As-impacted soils were used to evaluate the effects of phosphate on As(V) reduction and As(III) oxidation. Cultures were initially spiked with various P:As ratios, incubated for approximately 48 h, and analyzed periodically for As(V) and As(III) concentration. Arsenate reduction was inhibited at high P:As ratios and completely suppressed at elevated levels of phosphate (500 and 1,000 μM; P inhibition constant (K(i))～20-100 μM). While high P:As ratios effectively shut down microbial As(V) reduction, the expression of the arsenate reductase gene (arsC) was not inhibited under these conditions in the As(V)-reducing isolate, Agrobacterium tumefaciens str. 5B. Further, high phosphate ameliorated As(V)-induced cell growth inhibition caused by high (1mM) As pressure. These results indicate that phosphate may inhibit As(V) reduction by impeding As(V) uptake by the cell via phosphate transport systems or by competitively binding to the active site of ArsC.     

Interactions of arsenate, sulfate and phosphate with yeast mitochondria

In the presence of K(+), addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (DeltaPsi) and this is prevented by millimolar concentrations of phosphate (PO(4)). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO(4)) and sulfate (SO(4)) were as efficient as PO(4) to protect mitochondria against the K(+) mediated swelling, depletion of the DeltaPsi, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO(4), SO(4) or AsO(4) was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO(4) and AsO(4) on the F(1)F(0)-ATPase were tested: both SO(4) and AsO(4) inhibited the synthesis of ATP following competitive kinetics against PO(4) and non-competitive kinetics against ADP. The mersalyl sensitive uptake of (32)PO(4) was not inhibited by SO(4) or AsO(4), suggesting that the synthesis of ATP was inhibited at the F(1)F(0)-ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO(4) or sulfite (SO(3)) were added. It is suggested that the structure and charge similarities of PO(4), AsO(4) and SO(4) result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K(+); at the second site, in the F(1)F(0)-ATPase, AsO(4) and SO(4) compete for binding against PO(4) leading to inhibition of the synthesis of ATP.     

Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L.

Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670, or 5340 mol kg^–1 arsenate and 800, 1600, or 3200 mol kg^–1 phosphate, respectively. Interactions between arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and phosphate. At low and medium arsenate levels (670 and 2670 mol kg^–1), phosphate had slight effects on arsenate uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 mol kg^–1). Moderate doses of arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.     

Accumulation of phosphate, sulfate and sucrose by excised Phloem tissues

Excised petiolar vascular bundles and excised phloem tissues have been shown to take up phosphate, sulfate and sucrose by a true accumulation process and against high concentration ratios. Phosphate was accumulated principally as inorganic phosphate, and sucrose principally as sucrose. The rates of accumulation of the 3 solutes into the phloem-containing tissues were from 4 to 35 times higher than into comparable parenchyma tissue. It is suggested that this active accumulation mechanism plays an important role in the phenomenon of phloem transport.

The excised vascular, phloem and parenchyma tissues show an aging phenomenon: aerating the excised tissues for 18 hours prior to their use causes marked changes in the accumulatory behavior of the tissue. The data suggest that 1 phosphate accumulation system of low affinity but high capacity exists in fresh tissue, and that aging allows the development of a second, additional phosphate accumulation mechanism of high affinity and low capacity. A possible role in the control of phosphate movement is suggested.

     

Effect of arsenate on inorganic phosphate transport in Escherichia coli.

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.     

Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [¹⁴C]carbamoyl aspartate from [¹⁴C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.     

The distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.     

Interaction of arsenate with phosphate-transport systems in wild- type and mutant Streptococcus faecalis

Harold, F. M. (National Jewish Hospital, Denver, Colo.), and J. R. Baarda. Interaction of arsenate with phosphate-transport systems in wild-type and mutant Streptococcus faecalis. J. Bacteriol. 91:2257-2262. 1966.-Arsenate competitively inhibits the growth of Streptococcus faecalis, primarily by competition with phosphate for a common transport system. Arsenate is itself accumulated by the cells; the uptake requires metabolic energy, and the intracellular arsenate level may reach 0.01 m. Cells loaded with arsenate have lost the capacity to take up radioactive glutamate, rubidium, phosphate, or arsenate itself, apparently by the uncoupling of adenosine triphosphate generation. The pH dependence of arsenate uptake is complex. At low concentrations of extracellular arsenate, uptake by the wild-type strain 9790 exhibits a single maximum about pH 8; mutant PT-1, previously shown to be defective in phosphate uptake, takes up essentially no arsenate. At high concentrations of arsenate, uptake by the wild type is bimodal with maxima at pH 5.5 and 9; the uptake curve for mutant PT-1 corresponds to the shoulder in the curve for the wild type. The apparent dissociation constant for arsenate uptake by the wild type is approximately 10(-5)m from pH 5 to 9, whereas that for mutant PT-1 is about 5 x 10(-5) M at pH 5 and rises rapidly with increasing pH. The results confirm the earlier conclusion that the lesion in mutant PT-1 resides in the transport of phosphate and arsenate. It is proposed that the wild type has two distinct transport systems, whereas the mutant has lost the one with alkaline pH optimum.     

A study of the release of phosphate and arsenate from yeast

Under certain conditions yeast cells release previously accumulated phosphate or arsenate. This efflux occurs only when metabolism is not inhibited. There is a rough correlation between the content of orthophosphate and the rate of release.     

The interaction of pyridoxal phosphate with aspartate apoaminotransferase

The rate of binding of pyridoxal phosphate to the apoenzyme of pig heart cytoplasmic aspartate aminotransferase (l-aspartate: 2-oxoglurate aminotransferase, EC 2.6.1.1) was measured by absorption spectroscopy and by formation of active enzyme. At pH 5.1 and 8.3 the binding of coenzyme follows saturation kinetics. The binding process thus involves at least two steps.The rate of pyridoxal phosphate binding to the apoenzyme is dependent on the anion present in the pH 8.3 triethanolamine buffer. Chloride activates somewhat at very low concentrations. Phosphate and its methyl, ethyl, and phenyl esters are very effective inhibitors of the recombination in that 0.2–0.4 mM inhibit the rate of coenzyme binding by 50%. This is below the physiological concentration of phosphate. Sulfate also inhibits the rate of binding, but nitrate and acetate have little effect.