A recombinant tyrosine aminotransferase from Trypanosoma cruzi has both tyrosine aminotransferase and alanine aminotransferase activities

Abstract Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-trans-ferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent K m values similar to those for the natural enzyme, thus proving that they belong to the same protein.     

O-acetylserine (thiol) lyase activity in Phragmites and Typha plants under cadmium and NaCl stress conditions and the involvement of ABA in the stress response

The roles of O-acetylserine (thiol) lyase (OASTL, EC 4.2.99.8) and abscisic (ABA) acid in stress responses to NaCl and cadmium treatments were investigated in Typha latifolia L. and Phragmites australis (Cav.) Trin. ex Steudel plants. OASTL activity increased under stress (25-300 microM Cd, 100mM NaCl, 1 microM ABA) in both Typha and Phragmites mainly in roots, contributing substantially to satisfy the higher demand of cysteine for adaptation and protection. The earliest significant responses in intact roots were recorded after 12-24 h of Cd treatments, but different levels of stimulation were also observed after 3 and 7 days of exposure. The OASTL activity responses of Phragmites to salinity (100mM NaCl) were higher than those of Typha. Cysteine synthesis in Typha is much higher than in Phragmites, which supports the efficiency of the thiol-metabolism-based protection shown in Typha. Exogenous ABA increased OASTL activity in both species. Cd treatments led to increased ABA levels in roots. Phragmites showed higher ABA levels compared to Typha. The increase of ABA content indicates the involvement of this phytohormone in early stress responses, while the stimulation of OASTL following the ABA application suggests that ABA has a role in an OASTL activation pathway.     

Tocopherol quinone content of green algae and higher plants revised by a new high-sensitive fluorescence detection method using HPLC--effects of high light stress and senescence

A rapid, sensitive fluorescence method was applied here for detection of oxidized tocopherol quinones in total plant tissue extracts using HPLC, employing a post-column reduction of these compounds by a Zn column. Using this method, we were able to detect both alpha- and gamma-tocopherol quinones in Chamydomonas reinhardii with a very high degree of sensitivity. The levels of both compounds increased under high light stress in the presence of pyrazolate in parallel to a decrease in the content of the corresponding tocopherols. The formation of tocopherol quinones from tocopherols was apparently due to their oxidation by singlet oxygen, which is formed in photosystem II under high light stress. alpha-Tocopherol quinone was also detected in a variety of higher plants of different age, and its level was found to increase during senescence in leaves grown under natural conditions. In contrast to alpha-tocopherol quinone, gamma-tocopherol quinone was not found in the higher plant species investigated with the exception of young runner bean leaves, where the levels of both compounds increased dramatically during cold and light stress. Taking advantage of native fluorescence of the reduced alpha-tocopherol quinone (alpha-tocopherol quinol), it can be detected in plant tissue extracts with a high sensitivity. In young runner bean leaves, alpha-tocopherol quinol was found at a level similar to alpha-tocopherol.     

Slr0077 of Synechocystis has cysteine desulfurase as well as cystine lyase activity

NifS-like proteins activate sulfur for a variety of biosynthetic purposes. The genome of the cyanobacterium Synechocystis contains 4 nifS-related sequences of which only the slr0077 gene seems to be essential. In this report the heterologous production of the Slr0077 protein, its purification, and catalytic properties are described. Slr0077 produces alanine as well as pyruvate from cyst(e)ine as substrate; the product ratio depends on the redox conditions. Alanine is the typical product of orthodox NifS proteins, pyruvate formation is typical of the cystine lyase of Synechocystis which is the most peculiar member of the NifS protein family. The specific activities of Slr0077 for both reaction types are low as compared to the prototypic enzymes. Upon reaction with thiol-alkylating agents Slr0077 is not readily inactivated unlike NifS. The unique properties of Slr0077 add to the emerging picture that the NifS family of proteins comprises enzymes with a variety of distinct reactivities.     

Modification of reproductive development in Arabidopsis thaliana under spaceflight conditions

Reproductive development in Arabidopsis thaliana (L.) Heynh. cv. Columbia plants was investigated under spaceflight conditions on shuttle mission STS-51. Plants launched just prior to initiation of the reproductive phase developed flowers and siliques during the 10-d flight. Approximately 500 flowers were produced in total by the 12 plants in both the ground control and spaceflight material, and there was no significant difference in the number of flowers in each size class. The flower buds and siliques of the spaceflight plants were not morphologically different from the ground controls. Pollen viability tests immediately post-flight using fluorescein diacetate indicated that about 35% of the pollen was viable in the spaceflight material. Light-microscopy observations on this material showed that the female gametophytes also had developed normally to maturity. However, siliques from the spaceflight plants contained empty, shrunken ovules, and no evidence of pollen transfer to stigmatic papillae was found by light microscopy immediately post-flight or by scanning electron microscopy on fixed material. Short stamen length and indehiscent anthers were observed in the spaceflight material, and a film-like substance inside the anther that connected to the tapetum appeared to restrict the release of pollen from the anthers. These observations indicate that given appropriate growing conditions, early reproductive development in A. thaliana can occur normally under spaceflight conditions. On STS-51, reproductive development aborted due to obstacles in pollination or fertilization.     

An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana

Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein. The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants. We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsis thaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins. Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate characteristic of all bifunctional DNA glycosylases/lyases. Consistent with its in vitro activity, expression of AtOGG1 suppresses the mutator phenotype of an E. coli strain deficient in 8-oxoG repair. Our results suggest that AtOgg1 is an structural and functional homologue of Ogg1 and establish the presence of two distinct 8-oxoG repair enzymes in Arabidopsis.     

Properties of partially purified leaf ornithine aminotransferase of Brassica juncea under salt stress

Properties of the partially purified L-ornithine: 2-oxoacid aminotransferase (EC 2.6.1.13) of leaves of Brassica juncea salt tolerant somaclone SR3P6-2 and its parent cv. Prakash were studied. The enzyme from the somaclone SR3P6-2 was relatively more efficient in terms of its Km, Vmax, and Ea (activation energy) and required higher levels of chlorides for inhibition as compared to the enzyme from the parent cv. Prakash. These results suggest some salt-stress related changes in the enzyme.     

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.     

Glucocorticoid induction of tyrosine aminotransferase in kidney cortex

It has recently been reported that the glucocorticoid receptors present in kidney occur as two distinct forms which are segregated in the cortex and the medulla. We were interested in determining if glucocorticoid induction of the enzyme tyrosine aminotransferase (L-tyrosine: 2 oxoglutarate aminotransferase, E.C.2.6.1.5) also differed in these two areas of the kidney. Administration of the synthetic glycocorticoid, dexamethasone, resulted in a 2-fold induction of tyrosine aminotransferase in kidney cortex of adrenalectomized rats and no induction of the enzyme in kidney medulla. Examination of this response in rat brain revealed no induction of the enzyme by dexamethasone in this tissue.     

Phenylalanine ammonia lyase fromSporidiobolus pararoseus andRhodosporidium toruloides: Application for phenylalanine and tyrosine deamination

Simultaneous depletion of phenylalanine and tyrosine by phenylalanine ammonia lyase is described in a mutual competitive inhibition model. The enzymes obtained fromSporidiobolus pararoseus andRhodosporidium toruloides were charaterized in terms of stability, optimal reaction parameters and kinetic behaviour. Both enzymes followed Michaelis-Menten kinetics with respect to the two amino acids. However, the enzyme fromRhodosporidium toruloides was inhibited by high tyrosine concentrations.     

Tocopherol content and enzymatic antioxidant activities in chloroplasts from NaCl-stressed tomato plants

Changes in tocopherol, chlorophyll and TBARS levels and the activities of antioxidant enzymes i.e., GSH-Px, GST, and SOD in chloroplasts of tomato plants subjected to moderate (50 mM) and severe (150 mM) NaCl stress were determined. Increase in tocopherol content around the second day under both stresses did not correlate with the chlorophyll degradation while such correlation was observed from the fifth day of severe stress. The activities of GSH-Px and GST as well as TBARS content showed NaCl-induced enhancement which was dose- and time-dependent. However, chloroplastic SOD was rather not involved in the response of tomato plants to NaCl stress. The obtained results suggest that under the moderate stress similarly as in the early phase of severe stress tocopherol functions as a typical antioxidant, while in the late phase of the latter it may be involved in senescence signaling pathway and enables the recovery and recycling of the compounds significant for a plant organism.     

罗汉果色氨酸转氨酶基因SgTAR2的克隆及表达分析

色氨酸转氨酶基因家族,是直接参与植物生长素生物合成途径的关键酶基因。该研究在罗汉果转录组测序的基础上,结合RACE技术克隆罗汉果色氨酸转氨酶基因SgTAR2的全长cDNA序列和DNA序列;并对其进行生物信息学分析和时空表达分析。结果表明:克隆所得SgTAR2的cDNA全长序列2078bp,最长ORF为1332bp,编码443个氨基酸,Gen Bank登录号为KU949381,其编码蛋白具有2个蒜氨酸酶保守结构域和多个5'-PLP结合位点,推测其可能参与催化色氨酸转氨基作用、化学防御作用、生长素生物合成等生物学过程;SgTAR2基因DNA长为4103bp,含有4个内含子和5个外显子,其内含子具有多个高水平转录调控因子和多个与激素、环境等胁迫响应相关的作用元件,暗示SgTAR2基因内含子协同调控罗汉果生长素合成、抗胁迫反应、形态发育等生物学过程。实时荧光定量结果显示,SgTAR2基因在罗汉果各组织器官均有表达,在雌蕊和15d幼果期表达量较高,暗示该基因参与罗汉果果实早期发育。该研究结果表明SgTAR2参与了生长素介导的罗汉果不同生长发育过程,特别对幼果及花的起始发育和器官形态建成等具有重要意义。     

Evidence of a role for tyrosine dephosphorylation in the control of postgermination arrest of development by abscisic acid in Arabidopsis thaliana L

In the present paper evidence is presented indicating that tyrosine dephosphorylation is a key regulatory mechanism in postgermination arrest of Arabidopsis thaliana L. seed development mediated by abscisic acid (ABA). By using phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, the sensitivity to the inhibitory effect of ABA on seed germination is enhanced. Consistent with this finding, we demonstrate that the ABA-responsive gene, RAB18, is hyperinduced in seeds imbibed in ABA plus PAO, compared with seeds imbibed only with ABA.     

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

Two translesion synthesis DNA polymerase genes, AtPOLH and AtREV1, are involved in development and UV light resistance in Arabidopsis

Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.     

Action of the phosphonic analogue of tyrosine and of other tyrosine derivatives on rat liver tyrosine aminotransferase

Summary Tyrosine transamination has been investigatedin vitro with a preparation of rat liver tyrosine aminotransferase in the presence of several structural derivatives of the substrate, including the phosphonic analogue. The transamination by tyrosine aminotransferase (TAT) needs the presence in the substrate molecule of free amino and carboxylic groups, a three-carbon aliphatic chain, a para-phenolic hydroxylic function and al-configuration. Some tyrosine analogues can markedly disturb the Tyr-TAT association: the chief structural modifications are (i) the removal of the free amine function in a compound still possessing a para-hydroxylic and a carboxylic group, (ii) the change of the carboxylic function by another acidic group, especially a phosphonic one, (iii) a disubstitution in positions 3 and 5. In every situation, the presence of a parahydroxylic group is compulsory to observe an inhibitory effect.     

Cellulose orientation at the surface of the Arabidopsis seedling. Implications for the biomechanics in plant development

In diffuse growing cells the orientation of cellulose fibrils determines mechanical anisotropy in the cell wall and hence also the direction of plant and organ growth. This paper reports on the mean or net orientation of cellulose fibrils in the outer epidermal wall of the whole Arabidopsis plant. This outer epidermal wall is considered as the growth-limiting boundary between plant and environment. In the root a net transverse orientation of the cellulose fibrils occurs in the elongation zone, while net random and longitudinal orientations are found in subsequent older parts of the differentiation zone. The position and the size of the transverse zone is related with root growth rate. In the shoot the net orientation of cellulose fibrils is transverse in the elongating apical part of the hypocotyl, and longitudinal in the fully elongated basal part. Leaf primordia and very young leaves have a transverse orientation. Throughout further development the leaf epidermis builds a very complex pattern of cells with a random orientation and cells with a transverse or a longitudinal orientation of the cellulose fibrils. The patterns of net cellulose orientation correlate well with the cylindrical growth of roots and shoots and with the typical planar growth of the leaf blade. On both the shoot and the root surface very specific patterns of cellulose orientation occur at sites of specific cell differentiation: trichome-socket cells complexes on the shoot and root hairs on the root.