Reconstitution of water channel function and 2D-crystallization of human aquaporin 8

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3?.     

Yeast methionine aminopeptidase I. Alteration of substrate specificity by site-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine). Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine. Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln356 (Gln233 in E. coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan. Mutation of Ser195 to alanine had no effect on substrate specificity. None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.     

Receptors for chemotaxis in Bacillus subtilis.

At least three receptors for chemotaxis toward L-amino acids in Bacillus subtilis could be found with the aid of taxis competition experiments. They are called the asparagine receptor, which detects asparagine and glutamine, the isoleucine receptor, which detects isoleucine, leucine, valine, phenylalanine, serine, threonine, cysteine, and methionine, and the alanine receptor, which detects alanine and proline. Histidine and glycine could not be assigned to one of these receptors. Cysteine and methionine were found to be general inhibitors of chemotaxis and serine was found to be a general stimulator of chemotaxis. Some structural analogues of amino acids were tested for chemotactic activity. The chemotactic activity of B. subtilis is compared with that of Escherichia coli.     

Changes in free amino acids and polyamine levels in Satsuma leaves in response to Asian citrus psyllid infestation and water stress

The effects of biotic and abiotic stresses on changes in amino acids and polyamine levels in Satsuma orange （Citrus unshiu; cultivar Owari） leaves were inves- tigated. Asian citrus psyllids Diaphorina citri （Kuwayama） （ACP） infestation was used to induce biotic stress while a water deficit was imposed to induce abiotic stress. Potted trees were infested by placing 50 psyllids on 3 citrus leaves enclosed in nylon mesh bags for 5 d. A parallel set of plants were kept water stressed by maintaining the soil at 20% water holding capacity for 5 d. Levels of total free amino acids were higher in water stressed and ACP infested leaves. Polyamine putrescine increased in infested leaves but not in water stressed leaves. Proline was the most abundant amino acid and its levels significantly increased by both biotic and abiotic stresses. Proline levels in infested leaves were significantly higher than the water stressed leaves. Histidine, methionine, asparagine, arginine, serine, and leucine levels also increased significantly in infested leaves, but in water stressed leaves only leucine, methionine, and threonine increased. Levels of amino acids, such as tyrosine, isoleucine, phenylalanine, glutamic acid, and alanine, declined in infested leaves. Under water stress asparagine, phenylalanine, serine, and histidine also declined compared to controls. This indicates that while proteolysis occurred under both stresses, metabolic conversion of amino acids was different under the two stresses. In ACP infested leaves some amino acids may be used as feeding material and/or converted into secondary metabolites for defense.     

Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood-Brain Transport of Leucine and Phenylalanine

The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+ = 2.5 mmol X kg-1 X h-1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.     

Improvement of Vero cell growth in glutamate-based culture by supplementing ammoniagenic compounds

Ammonia has been identified as one of the most inhibitory substances for mammalian cells. We have attempted to develop a less-ammoniagenic medium for the growth of Vero cells by substitution of glutamine with glutamate. In spite of reduced ammonia formation, Vero cells cultured in glutamate-based medium (DMEM-glu) could not grow normally as in glutamine-based medium (DMEM-gln). After Vero cells adapted to DMEM-glu, alanine was consumed instead of accumulated and both asparagine and glutamine were almost undetectable, indicating the lacking for aminonitrogen. By supplementing NH₄Cl, the growth was significantly improved and the cellular uptake of glutamate from medium was greatly increased. However the growth was still not restored to the level in DMEM-gln, likely due to ammonia toxicity. Asparagine was chosen to support the growth of Vero cells in DMEM-glu, formulating DMEM-glu-asn. This replacement reduced ammonia formation by 79% and increased cell yields by 34% compared with DMEM-gln. After Vero cells adapted to DMEM-glu-asn, glutamine synthetase (GS) activity was elevated by 3.8 folds compared with control in DMEM-gln. In DMEM-glu-asn Vero cell growth was arrested by the specific GS inhibitor, methionine sulphoximine. This arrest affirmed the essential role of GS in glutamine synthesis and disconfirmed the potential role of asparagine synthase (AS) in glutamine formulation (also asparagine utilization).     

Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.     

Utilization of Nitrogen Sources by Immature Soybean Cotyledons in Culture

Immature Glycine max (L.) Merrill cotyledons were cultured ina defined medium containing different nitrogen sources. Glutaminewas the most efficient source in terms of protein accumulationin the cotyledons. Asparagine was less efficient (about 70 percent that of glutamine) while allantoin was a poor source ofnitrogen. This was also true for older cotyledons where asparaginaseand allantoinase activities were maximal. The utilization ofboth asparagine and allantoin (but not glutamine) was totallyinhibited by methionine sulfoximine suggesting that their metabolisminvolves ammonia assimilation via glutamine synthetase. Apparently,neither exogenous or endogenously-generated ammonia had mucheffect on glutamine utilization, but ammonia did have a smallinhibitory effect on asparagine, which may in part account forthe lower efficiency observed with this amide. Glycine max, soybean, cotyledon culture, nitrogen metabolism     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Aquaporin-8-facilitated mitochondrial ammonia transport

Aquaporin-8 (AQP8) is a membrane channel permeable to water and ammonia. As AQP8 is expressed in the inner mitochondrial membrane of several mammalian tissues, we studied the effect of the AQP8 expression on the mitochondrial transport of ammonia. Recombinant rat AQP8 was expressed in the yeast Saccharomyces cerevisiae. The presence of AQP8 in the inner membrane of yeast mitochondria was demonstrated by subcellular fractionation and immunoblotting analysis. The ammonia transport was determined in isolated mitochondria by stopped flow light scattering using formamide as ammonia analog. We found that the presence of AQP8 increased by threefold mitochondrial formamide transport. AQP8-facilitated mitochondrial formamide transport in rat native tissue was confirmed in liver (a mitochondrial AQP8-expressing tissue) vs. brain (a mitochondrial AQP8 non-expressing tissue). Comparative studies indicated that the AQP8-mediated mitochondrial movement of formamide was markedly higher than that of water. Together, our data suggest that ammonia diffusional transport is a major function for mitochondrial AQP8.     

The terminal amino acids of protein sequences and protein maturation

A comparison is made of the N- and C-terminal amino acids from 96 published protein sequences, 26 from prokaryotes, 70 from eukaryotes. The observed frequencies of the N-terminal amino acids methionine, alanine and serine in prokaryotes, and alanine and serine in eukaryotes are significantly higher than expected for a random arrangement of amino acids. At the C-terminal end, the observed frequencies of lysine, asparagine and glutamine in prokaryotes and phenylalanine, asparagine and glutamine in eukaryotes exceed random expectation. These results could be explained by specific proteolytic cleavage during protein synthesis.     

谷氨酰胺和天冬酰胺对CHO细胞生长、代谢及抗体表达的影响

以离心换液的批培养为例,通过设计谷氨酰胺和天冬酰胺不同的添加方式来考察两者对CHO细胞生长,代谢及产物表达的影响。结果表明：基础培养基中谷氨酰胺和天冬酰胺不能简单地相互替换,缺失谷氨酰胺或天冬酰胺的基础培养基均不能支持dhfr-CHO细胞的正常生长和产物表达,仅谷氨酰胺和天冬酰胺的浓度同时达到4mmol/L,才能满足细胞生长所需。另外,代谢副产物氨的生成仅与谷氨酰胺和天冬酰胺的加和线性相关,与两者添加比例无关。但适当提高天冬酰胺与谷氨酰胺的比例可提高抗体表达水平,同时减少乳酸的生成。因此,为培养基开发与优化过程中谷氨酰胺和天冬酰胺的添加策略提供了依据,为建立高效的流加培养过程奠定了基础。     

Active water selective channels in the stomach: investigation of aquaporins after ethanol and capsaicin treatment in rats

Recent studies discovered the existence of aquaporins (AQP), suggesting their roles in the active, ATP dependent water secretion or absorption. Our recent development of the monoclonal antibody family against aquaporins (Type 1 and 4) allowed us a good opportunity to investigate the mechanism of the gastric mucosal edema in a rat model. THE AIM OF OUR STUDY was to evaluate the changes in the tissue level of aquaporins (AQP1 and AQP4) after ethanol and capsaicin treatment in rat stomach. MATERIALS AND METHODS: the experiments were carried out on Sprague-Dawley rats weighing 150-200 g. The animals were fasted for 24 h, after the 1 ml of ethanol (50% v/v) or capsaicin (2 mg/ml) was given intragastrically. Rats were sacrificed after 5, 30, 60, 120 and 240 min, the tissue level of AQP1 and AQP4 was investigated immunoserologically by ELISA and dot-blot methods using our monoclonal antibodies. The location of these aquaporins in the gastric tissue was demonstrated by immunohistochemistry. RESULTS: (1) in ethanol-treated stomach, both AQP1 and AQP4 increased after 5 min simultaneously with gastritis, then decreased dramatically depending on time. (2) In the capsaicin-treated group there were no changes in the tissue level of aquaporins in the first hour. After 60 min both AQP1 and AQP4 increased in the stomach without any macroscopically detectable changes, then decreased depending on time. (3) The immunohistochemical investigations using our monoclonal antibodies seem to support our present quantitative results. CONCLUSION: chemically induced gastric mucosal lesions are started by an extended edema. In the induction of the edema and the subsequent gastric injury, aquaporins (both AQP1 and AQP4) play an important role in the maintenance of mucosal integrity.     

Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase

Amino-terminal processing in the yeast Saccharomyces cerevisiae has been investigated by examining numerous mutationally altered forms of iso-1-cytochrome c. Amino-terminal residues of methionine were retained in sequences having penultimate residues of arginine, asparagine, glutamine, isoleucine, leucine, lysine, and methionine; in contrast, the amino-terminal methionine residues were exercised from residues of alanine, glycine, and threonine and were partially excised from residues of valine. The results suggest the occurrence of a yeast aminopeptidase that removes amino-terminal residues of methionine when they precede certain amino acids. A systematic search of the literature for amino-terminal sequences formed at initiation sites suggests the hypothetical yeast aminopeptidase usually has the same specificity as the amino peptidase from bacteria and higher eukaryotes. Our results and the results from the literature search suggest that the aminopeptidase cleaves amino-terminal methionine when it precedes residues of alanine, glycine, proline, serine, threonine, and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine glutamic acid, isoleucine, leucine, lysine, or methionine. In contrast to the normal iso-1-cytochrome c and in contrast to the majority of the mutationally altered proteins, certain forms were acetylated including the following sequences: acetyl(Ac)-Met-Ile-Arg-, Ac-Met-Ile-Lys, Ac-Met-Met-Asn-, and Ac-Met-Asn-Asn-. We suggest yeast contains acetyltransferases that acetylates these mutant forms of iso-1-cytochromes c because their amino-terminal regions resemble the amino-terminal regions of natural occurring proteins which are normally acetylated. The lack of acetylation of closely related sequences suggest that the hypothetical acetyltransferases are specific for certain amino-terminal sequences and that the 3 amino-terminal residues may play a critical role in determining these specificities.     

A convenient gHMQC-based NMR assay for investigating ammonia channeling in glutamine-dependent amidotransferases: studies of Escherichia coli asparagine synthetase B

X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformation have revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels, giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficiently into a separate synthetase site where it undergoes further reaction. We now report a very convenient isotope-edited 1H NMR-based assay that can be used to probe the transfer of ammonia between the active sites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B (AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solution can react with the thioester intermediate formed during the glutaminase half-reaction by accessing the N-terminal active site of AS-B during catalytic turnover. These observations are consistent with a model in which exogenous ammonia can access the intramolecular tunnel in AS-B during glutamine-dependent asparagine synthesis, in contrast to expectations based on studies of class I amidotransferases.     

Molecular determinants of ammonia and urea conductance in plant aquaporin homologs

Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP-like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP-like mutants conducted urea but not NH(3), and a residue without direct contact to the pore lumen was critical for conduction in the mutants.