Solvent Extraction of Li+ using Organophosphorus Ligands in the Presence of Ammonia

In this work, the selective extraction of Li⁺ with the aid of organophosphorus ligands (H-OP) including phenylphosphonic (H-PHO), phenylphosphinic (H-PHI) and bis(2-ethylhexyl) phosphoric (H-BIS) acids in the absence and presence of ammonia was studied. Adding NH₃ to the aqueous phase resulted in significant improvement in the % extraction of Li⁺ into the organic phase containing H-OP ligands. The highest % extraction values obtained in the case of H-PHO, H-PHI, and H-BIS were 43.2%, 45.7%, and 90.0%, respectively. Two mechanisms were inferred; the first was that the extraction equilibrium reaction of LiCl + H-OP ? Li-OP + HCl shifted forward due the reaction of the produced HCl with NH₃. The second mechanism was that the Li⁺/NH₄⁺ exchange of NH₄-OP (produced from the reaction of H-OP with NH₃) was easier than Li⁺/H⁺ exchange of H-OP itself. Competitive extraction experiments indicated that the selectivity factors of Li⁺ over Na⁺ and K⁺ were strongly dependent on the concentration of H-OP ligands which suggested that aggregation of ligand molecules via hydrogen bonding is the limiting factor for selectivity.     

Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H⁺ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H⁺ transport activity, namely, H⁺-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H⁺ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H⁺-ATPases mediate H⁺ extrusion, whereas most members of other six proteins mediate H⁺ influx, thus contributing to cytosolic pH homeostasis by directly modulating H⁺ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H⁺-coupled substrate influx, whereas other intra-family members facilitate H⁺-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H⁺-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H⁺ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H⁺ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.     

Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm

Sperm-specific K⁺ ion channel (KSper) and Ca²⁺ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca²⁺ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pH_i) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na⁺/H⁺ exchangers (NHEs) have been implicated to mediate pH_i in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N,N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pH_i acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca²⁺, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.     

Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser⁷⁸⁵ and Ser⁷⁸⁷, that were similar in location and context to two amino acids of the Arabidopsis Na⁺/H⁺ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.     

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa

H⁺/K⁺ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific β subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the β₁ subunit of Na⁺/K⁺ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.     

Cloning,Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae)

The vacuolar (H⁺)-ATPase (V-ATPase) of insect, which is composed of membrane-bound V₀ complex and peripheral V₁ complex, participates in lots of important physiological process. Subunit H, as a subunit of V₁ complex, plays a vital role in bridging the communication between V₁ and V₀ complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends) technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.     

Roles of the Na+/H+ Exchanger Isoform 1 and Urokinase in Prostate Cancer Cell Migration and Invasion

Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na⁺/H⁺ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1—alone, or with inhibitors combining NHE1 or uPA inhibition—generally did not prevent prostate cancer cell migration. However, uPA inhibition—but not NHE1 inhibition—prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo.     

Prokaryotic Na+/H+ Exchangers—Transport Mechanism and Essential Residues

Na⁺/H⁺ exchangers are essential for Na⁺ and pH homeostasis in all organisms. Human Na⁺/H⁺ exchangers are of high medical interest, and insights into their structure and function are aided by the investigation of prokaryotic homologues. Most prokaryotic Na⁺/H⁺ exchangers belong to either the Cation/Proton Antiporter (CPA) superfamily, the Ion Transport (IT) superfamily, or the Na⁺-translocating Mrp transporter superfamily. Several structures have been solved so far for CPA and Mrp members, but none for the IT members. NhaA from E. coli has served as the prototype of Na⁺/H⁺ exchangers due to the high amount of structural and functional data available. Recent structures from other CPA exchangers, together with diverse functional information, have allowed elucidation of some common working principles shared by Na⁺/H⁺ exchangers from different families, such as the type of residues involved in the substrate binding and even a simple mechanism sufficient to explain the pH regulation in the CPA and IT superfamilies. Here, we review several aspects of prokaryotic Na⁺/H⁺ exchanger structure and function, discussing the similarities and differences between different transporters, with a focus on the CPA and IT exchangers. We also discuss the proposed transport mechanisms for Na⁺/H⁺ exchangers that explain their highly pH-regulated activity profile.     

Inhibition of gastric H+, K+-ATPase by novel thiazolidinone derivatives

In a program to identify new anti-ulcer compounds, a series of novel substituted thiazolidinone derivatives 5(a–j) were synthesized and screened for their in vitro H⁺, K⁺-ATPase inhibitory activity. The synthesized compounds were characterized by nuclear magnetic resonance (¹H-NMR), liquid chromatography-mass spectrometry (LCMS) and fourier transform infrared (FTIR) analysis. We have briefly investigated the structure–activity relation (SAR) studies and reveal that the nature of position of the fluorine atom influences the anti-ulcer activity. Among the synthesized compounds 5b, 5c and 5e showed 4 and 10-fold higher H⁺, K⁺-ATPase activity when compared with those of other derivatives 5a, 5f, 5g and 5j, respectively. H⁺, K⁺-ATPase activity of 5b, 5c and 5e were comparable with those of known H⁺, K⁺-ATPase blocker lansoprazole which is a potential anti-ulcer drug.     

Cyclic voltammetry studies of TiO2 nanotube arrays electrode: Conductivity and reactivity in the presence of H and aqueous redox systems

In this paper, we use cyclic voltammetry to investigate the effect of protons on the conductivity and reactivity of TiO₂ nanotube array (NTA) electrodes in an aqueous redox system. The H⁺ ion can change the TiO₂ nanotube (anatase phase) surface states for electrons transfer. It can also act as an intermediate state for electron transfer to the acceptor species in the electrolyte surrounding the TiO₂. The higher the concentration of H⁺ ions in the aqueous electrolyte, the easier it is for the electrons transfer from the TiO₂ to the electrolyte oxidized species. The reduction is facile, with a similar reduction potential for various acceptor species, but re-oxidation is not possible. It is apparently an electrochemical reduction involving a single-proton transfer and single-electron transfer. Based on this conclusion, an electrode (PB/Au/TiO₂ NTAs) was fabricated by means of electrodeposition. The electrode used to detect hydrogen peroxide. The sensitivity of the detector is high, and its the detection limit can be as low as 100 nM.     

Extraction Equilibria of 4-Hydroxypyridine using Di (2-ethylhexyl) Phosphoric Acid

Extractions of 4-Hydroxpyridine (4HP) from aqueous solutions using Di(2-ethylhexyl)phosphoric acid (D2EHPA) as extractant in 1-octanol and kerosene were studied. The factors that affected the distribution coefficient (D), such as equilibrium pH, the concentration of D2EHPA, and the type of diluents were discussed. The interaction mechanism between 4HP and D2EHPA was validated by infrared spectroscopic analysis. D increased with the increase of the concentration of D2EHPA and peak values appeared at equilibrium pH = 3.6–5.0. D in the polar diluent (1-octanol) was much higher than those in the non-polar diluent (kerosene). The extraction reaction was found to be a proton-transfer process and D2EHPA mainly reacted through its –OH with –N– of 4HP. The apparent reactive extraction equilibrium constants K ₁₁ and K ₁₂ were obtained by fitting the experimental data of extraction equilibrium. By comparing calculated D values from the proposed model with the experimental ones, the accuracy of the proposed model was examined.     

Reactivity of disulfide peptide l-cystine with iodido (diethylenetriamine)platinum(II) and synthesis of products in the presence of an iodide ion

Substitution reactions of complex [PtI(dien)]⁺ (where, dien=diethylenetriamine ) with sulfur-containing peptide l-cystine has been studied in 1.0×10^?1 M aqueous perchlorate or acetate medium between 298≤T (K)≤323 and 2.30≤pH≤4.45 using a UV–visible spectrophotometer. Products obtained have characterized from their physico-chemical and spectroscopic methods at various pH and temperatures. From this characterization, products have indicated that [PtI(dien)]⁺ has formed a complex with l-cystine and acts as a bidentate ligand, through Pt–S bond at 2.30≤pH≤3.30 and through Pt–N and Pt–S bond of cystine in 3.95≤pH≤4.45. At 2.30≤pH≤3.30, ring opening and closing of dien have occurred at 308 and 323 K, respectively, and the same has happened at pH≥3.95. All reactions have followed the rate law – d[mixture]/dt=(k ₁+k ₂ [cystine]) [Pt(II)], where k ₂ denotes the second-order rate constant. Activation parameters E _a , Δ H ^# and Δ S ^# have been determined. Product formation and reversible and forward reaction rate constants have also been evaluated.     

The Mechanism of Energy Coupling in H+/Na+-Pumping Membrane Pyrophosphatase—Possibilities and Probabilities

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H⁺ and/or Na⁺ transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps. Detailed phylogenetic studies have revealed various subtypes of mPPases and suggested their roles in the evolution of the “sodium” and “proton” bioenergetics. This treatise focuses on the mechanistic aspects of the transport reaction, namely, the coupling step, the role of the chemically produced proton, subunit cooperation, and the relationship between the proton and sodium ion transport. The available data identify H⁺-PPases as the first non-oxidoreductase pump with a “direct-coupling” mechanism, i.e., the transported proton is produced in the coupled chemical reaction. They also support a “billiard” hypothesis, which unifies the H⁺ and Na⁺ transport mechanisms in mPPase and, probably, other transporters.     

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H⁺-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H⁺-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H⁺-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H⁺-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H⁺-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.     

Complexes in the Photocatalytic Reaction of CO(2) and H(2)O: Theoretical Studies

Complexes (H(2)O/CO(2), e-(H(2)O/CO(2)) and h(+)-(H(2)O/CO(2))) in the reaction system of CO(2) photoreduction with H(2)O were researched by B3LYP and MP2 methods along with natural bond orbital (NBO) analysis. Geometries of these complexes were optimized and frequencies analysis performed. H(2)O/CO(2) captured photo-induced electron and hole produced e-(H(2)O/CO(2)) and h(+)-(H(2)O/CO(2)), respectively. The results revealed that CO(2) and H(2)O molecules could be activated by the photo-induced electrons and holes, and each of these complexes possessed two isomers. Due to the effect of photo-induced electrons, the bond length of C=O and H-O were lengthened, while H-O bonds were shortened, influenced by holes. The infrared (IR) adsorption frequencies of these complexes were different from that of CO(2) and H(2)O, which might be attributed to the synergistic effect and which could not be captured experimentally.     

Studies on promising cell performance with H2SO4 as the catholyte for electrogeneration of Ag2+ from Ag+ in HNO3 anolyte in mediated electrochemical oxidation process

Electrochemical performance of a divided cell with electrogeneration of Ag²⁺ from Ag⁺ in 6 M HNO₃ anolyte has been studied with 6 M HNO₃ or 3 M H₂SO₄ as the catholyte. This work arose because in mediated electrochemical oxidation (MEO) processes with Ag(II)/Ag(I) redox mediator, HNO₃ is generally used as catholyte, which, however, produces NO _x gases in the cathode compartment. The performance of the cell with 6 M HNO₃ or 3 M H₂SO₄ as the catholyte has been compared in terms of (i) the acid concentration in the cathode compartment, (ii) the Ag⁺ to Ag²⁺ conversion efficiency in the anolyte, (iii) the migration of Ag⁺ from anolyte to catholyte across the membrane separator, and (iv) the cell voltage. Studies with various concentrations of H₂SO₄ catholyte have been carried-out, and the cathode surfaces have been analyzed by SEM and EDXA; similarly, the precipitated material collected in the cathode compartment at higher H₂SO₄ concentrations has been analyzed by XRD to understand the underlying processes. The various beneficial effects in using H₂SO₄ as catholyte have been presented. A simple cathode surface renewal method relatively free from Ag deposit has been suggested.