Mechanisms of ammonium transport, accumulation, and retention in ooyctes and yeast cells expressing Arabidopsis AtAMT1;1

Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Δψ-driven transport of through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture.     

Regulation of NH4+ transport by essential cross talk between AMT monomers through the carboxyl tails

Ammonium transport across plant plasma membranes is facilitated by AMT/Rh-type ammonium transporters (AMTs), which also have homologs in most organisms. In the roots of the plant Arabidopsis (Arabidopsis thaliana), AMTs have been identified that function directly in the high-affinity NH4+ acquisition from soil. Here, we show that AtAMT1;2 has a distinct role, as it is located in the plasma membrane of the root endodermis. AtAMT1;2 functions as a comparatively low-affinity NH4+ transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH4+ transport activity. Coexpressing these mutants along with wild-type AtAMT1;2 substantially reduced the activity of the wild-type transporter. A molecular model of AtAMT1;2 provides a plausible explanation for the dominant inhibition, as the C terminus of one monomer directly contacts the neighboring subunit. It is suggested that part of the cytoplasmic C terminus of a single monomer can gate the AMT trimer. This regulatory mechanism for rapid and efficient inactivation of NH4+ transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium.     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.     

Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants

We have cloned and characterized the first member of a novel family of ammonium transporters in plants: AtAMT2 from Arabidopsis thaliana. AtAMT2 is more closely related to bacterial ammonium transporters than to plant transporters of the AMT1 family. The protein was expressed and functionally characterized in yeast. AtAMT2 transported ammonium in an energy-dependent manner. In contrast to transporters of the AMT1 family, however, AtAMT2 did not transport the ammonium analogue, methylammonium. AtAMT2 was expressed more highly in shoots than roots and was subject to nitrogen regulation.     

Copper·Lys-Gly-His-Lys mediated cleavage of tRNA: Studies of reaction mechanism and cleavage specificity

The reactivity of [Cu²⁺·Lys-Gly-His-Lys-NH₂]²⁺ and [Cu²⁺·Lys-Gly-His-Lys]⁺ toward tRNA^Phe has been evaluated. The amidated and carboxylate forms of the copper peptides display complex binding behavior with strong and weak sites evident (, for the amide form; and , for the carboxylate form), while Cu²⁺(aq) yielded and . The time-dependence of the reaction of [Cu²⁺·Lys-Gly-His-Lys]⁺ and [Cu²⁺·Lys-Gly-His-Lys-NH₂]²⁺ with tRNA^Phe yielded k_obs ∼ 0.075 h⁻¹ for both complexes. HPLC analysis of the reaction products demonstrated guanine as the sole base product. Mass spectrometric data shows a limited number of cleavage fragments with product peak masses consistent with chemistry occurring at a discrete site defined by the structurally contiguous D and TΨC loops, and in a domain where high affinity magnesium centers have previously been observed to promote hydrolysis of the tRNA^Phe backbone. This cleavage pattern is more selective than that previously observed by Long and coworkers for nickel complexes of a series of C-terminally amidated peptides (Gly-Gly-His, Lys-Gly-His, and Arg-Gly-His), and may reflect variations in structural recognition and a distinct reaction path by the nickel derivatives. The data emphasizes the optimal positioning of the metal-associated reactive oxygen species, relative to scissile bonds, as a major criterion for development of efficient catalytic nucleases or therapeutics.     

Effect of clotrimazole on the pump cycle of the Na,K-ATPase

The effect of the antimycotic drug clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady-state fluorescence experiments with the electrochromic styryl dye RH421 were combined with those achieved by a pre-steady-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E₂ state, that acts as a “dead-end” side track of the pump cycle, where X stands for H⁺ or K⁺. Na⁺ binding, enzyme phosphorylation, and Na⁺ transport were not affected by CLT, and at high CLT concentrations of the enzyme remained active in the physiological transport mode. The presence of Na⁺ and K⁺ destabilized the inactivated form of the Na,K-ATPase.     

Alkyl dehydrogenation in iridium tri-cyclopentyl phosphines

The iridium cyclooctadiene complex incorporating a tricyclopentyl phosphine ligand (PCyp₃), Ir(η²:η²-C₈H₁₂)(PCyp₃)Cl, has been prepared. Removal of the chloride from this complex using in CH₂Cl₂/arene solvent results in dehydrogenation (C-H activation followed by β-H transfer) of one of the alkyl phosphine rings and formation of the complexes (X = H, F) which contain a hybrid phosphine-alkene ligand. These complexes are formed alongside another product (5-20% yield) that has been identified as , which can be prepared in high yield by an alternative, and slightly modified, route. This complex is with a minor isomer that has been tentatively identified as , which results from allylic C-H activation of cyclooctadiene. Addition of H₂ to and its isomer in arene solvent (C₆H₅X, X = F, H) forms the dihydrido η⁶-arene Ir(III) complexes . In contrast, hydrogenation in CH₂Cl₂ alone results in the formation of in which the anion is now acting as a ligand through one of its aryl rings. The fluorobenzene complex can be cleanly converted to by addition of the hydrogen acceptor tert-butylethene (tbe).     

Measuring and modeling chloride-hydroxyl exchange in the Guinea-pig ventricular myocyte

Protons are powerful modulators of cardiac function. Their intracellular concentration is regulated by sarcolemmal ion transporters that export or import H⁺-ions (or their ionic equivalent: ). One such transporter, which imports H⁺-equivalents, is a putative Cl⁻/OH⁻ exchanger (CHE). A strong candidate for CHE is SLC26A6 protein, a product of the SLC26A gene family of anion transporters, which has been detected in murine heart. SLC26A6 protein is suggested to be an electrogenic ) exchanger. Unfortunately, there is insufficient characterization of cardiac CHE against which the properties of heterologously expressed SLC26A6 can be matched. We therefore investigated the proton, Cl⁻, and voltage dependence of CHE activity in guinea-pig ventricular myocytes, using voltage-clamp, intracellular pH fluorescence, and mathematical modeling techniques. We find that CHE activity is tightly regulated by intracellular and extracellular pH, is voltage-insensitive over a wide range (±80 mV), and displays substrate dependence suggestive of electroneutral 1Cl⁻/1OH⁻ exchange. These properties exclude electrogenic SLC26A6 as sole contributor to CHE. Either the SLC26A6 product in heart is electroneutral, or CHE comprises at least two transporters with oppositely balanced voltage sensitivity. Alternatively, CHE may comprise an H⁺-Cl⁻ coinflux system, which cannot be distinguished kinetically from an exchanger. Irrespective of ionic mechanism, CHE's pH sensitivity helps to define resting intracellular pH, and hence basal function in the heart.     

Conformational isomerism and hydrogen-bonded motifs of anion assisted supramolecular self-assemblies using Cu/Co salts and pyridine-4-acetamide

Six novel metal-organic complex assemblies constructed from a conformation-flexible ligand - pyridine-4-acetamide (PAT) and inorganic Cu^II and Co^II salts have been synthesized and structurally characterized by single crystal X-ray diffraction analysis. Crystal structure analysis reveals five types of architectures by variation of metal salts. In {[Cu(PAT)₂Cl₂]}_n (1) and {[Co(PAT)₂Cl₂]}_n (3), PAT ligands bridge metal centers to form one-dimensional chains. The chains are extended to three dimensions with the aid of two types of hydrogen bonded motifs () and (12)). {[Cu(PAT)₂(NO₃)](NO₃)(THF)}_n (5) which exhibits two-dimensional coordinating layers forms open channels filled with solvent molecules. In [Cu(PAT)₂Cl₂] (2), [Co(PAT)₂Cl₂] (4) and [Co(PAT)₄(H₂O)₂](NO₃)₂(THF)₂ (6), PAT is observed as a monofunctional ligand. Complex 2 forms one-dimensional hydrogen bonded chains. Crystal structure of complex 4 has a two-dimensional infinite hydrogen-bonded network with and motifs formed by complementary amide-amide hydrogen bonds. [Co(PAT)₄(H₂O)₂](NO₃)₂(THF)₂ (6) crystallizes in centrosymmetric I4₁/a space group. Complex 6 forms chiral channels which are filled with twisted solvent helices and anion helices. Within each channel the solvent helix and the anion helix have the same handedness; and adjacent channels have opposite handedness. Complexes 1, 2 and complexes 3, 4 illustrate examples of conformational supramolecular isomerism in {[Cu(PAT)₂Cl₂]}_∞ and {[Co(PAT)₂Cl₂]}_∞, respectively. In these complexes, changes of PAT conformations and coordination geometry of metal center induced the structural versatility.     

Dinuclear gold(III) pyrazolato complexes - Synthesis, structural characterization and transformation to their trinuclear gold(I) and gold(I/III) analogues

The reaction of AuCl₃py with Na(pz∗) (pz∗ = pyrazolato, or substituted pyrazolato anion) yields stable dinuclear [cis-Au^IIICl₂(μ-pz∗)]₂ complexes. In the presence of a base, the latter undergo reduction with concomitant transformation of the dinuclear -structure to trinuclear Au^I, Au^III (containing trans Au^IIICl₂-centres) and species.     

Ruthenium(II) complexes possessing the η-p-cymene ligand

Complexes possessing a soft donor η⁶-arene and hard donor acetylacetonate ligand, [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1) (OTf = trifluoromethanesulfonate; acac = acetylacetonate) and {Ar′ = 3,5-(CF₃)-C₆H₃}, were prepared and fully characterized. The lability of the μ-CH linkage for complex 1 and the THF ligand of 2 allow access to the unsaturated cation [(η⁶-p-cymene)Ru(κ²-O,O-acac)]⁺. The reaction of with KTp {Tp = hydridotris(pyrazolyl)borate} produces . The azide complex forms upon reaction of with N₃Ar (Ar = p-tolyl), and reaction of with CHCl₃ at 100 °C yields the chloride-bridged binuclear complex . The details of solid-state structures of [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1), and are disclosed.     

CLC transport proteins in plants

Nitrate compartmentalization in intracellular organelles has been long recognized as critical for plant physiology but the molecular identity of the proteins involved remained unclear for a long time. In Arabidopsis thaliana, AtClC-a has been recently shown to be a antiporter critical for nitrate transport into the vacuoles. AtClC-a is a member of the CLC protein family, whose animal and bacterial members, comprising both channels and H⁺-coupled antiporters, have been previously implicated exclusively in Cl⁻ transport. Despite the different over Cl⁻ selectivity of AtClC-a compared to the other CLC antiporters, it has similar transport properties.Other CLC homologues have been cloned in Arabidopsis, tobacco, rice and soybean.     

Inhibition by purine nucleotides of the release of reactive oxygen species from muscle mitochondria: indication for a function of uncoupling proteins as superoxide anion transporters

Release of reactive oxygen species (ROS), measured as the sum of hydrogen peroxide (H₂O₂) and superoxide anion radical (), from respiring rat heart and skeletal muscle mitochondria was significantly decreased by millimolar concentrations of GTP or GDP. Attempts to differentiate between the two forms of ROS showed that the release of rather than that of H₂O₂ was affected. Meanwhile, intramitochondrial ROS accumulation, measured by inactivation of aconitase, increased. These results suggest that guanine nucleotides inhibit the release of from mitochondria. As these nucleotides are known inhibitors of uncoupling proteins (UCPs), it is proposed that UCPs may function as carriers of , thus enabling its removal from the matrix compartment.     

The crystal and molecular structures of dichlorobis (5,7-diphenyl-1,2,4-triazolo[1,5-α]pyrimidine)zinc(II) (1) and dichlorobis(5,7-diphenyl-1,2,4-triazolo[1,5-α]pyrimidine)cobalt(II) (2)

The synthesis and structural studies of the new ligand 5,7-diphenyl-1,2,4-triazolo[1,5-α]pyrimidine (dptp), which can be considered as an analog of purine, and its complexes are described. Complexes were characterised by spectral measurements (IR, NMR, UV-Vis). In addition X-ray structural analysis was performed. Crystals of [Zn(C₁₇H₁₂N₄)₂Cl₂] (1) revealed the following parameters: M_r = 680.9; monoclinic for 2188 observed reflections. [Co(C₁₇H₁₂N₄)₂Cl₂] (2); M_r = 674.4; monoclinic for 1630 observed reflections.