Withania somnifera （Ashwagandha）： a Novel Source of L-asparaginase

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme （E.C.3.5.1.1.）. Among 34 plant species screened for L-asparaginase enzyme. Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 ℃. The Km value for the enzyme is 6.1 × 10^-2 mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant.     

Verticase： a Fibrinolytic Enzyme Produced by Verticillium sp, Tj33, an Endophyte of Trachelospermum jasminoides

Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of VerticUlium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pl of 8.5, was demonstrated to be homogeneous by sodium dodecyl suIfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9-10 and 50-60℃, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease.     

Functional expression and purification of cytokinin dehydrogenase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (AtCKX2) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 μkat g⁻¹, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Expression and Antigenic Characterization of the Epitope-G1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

The epitope-G1 gene of Bovine ephemeral fever virus(BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1.Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115.The recombinant P.pastoris strains were selected by a G418 transformation screen and confirmed by PCR.After being induced with methanol,an expressed protein with 26 kDa molecular weight was obtained,which was much bigger than the predicted size(15.54 kDa).Deglycosylation analysis indicated the recombinant G1 was glycosylated.Western blot and ELISA tests,as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity.The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.     

Discovery and identification of Serum Amyloid A protein elevated in lung cancer serum

Two hundred and eighteen serum samples from 175 lung cancer patients and 43 healthy individuals were analyzed by using Surface Enhaced Laser Desorption/Ionization Time of Flight Mass Spectrome- try (SELDI-TOF-MS). The data analyzed by both Biomarker Wizard? and Biomarker Patterns? software showed that a protein peak with the molecular weight of 11.6 kDa significantly increased in lung cancer. Meanwhile,the level of this biomarker was progressively increased with the clinical stages of lung cancer. The candidate biomarker was then obtained from tricine one-dimensional sodium dodecyl sul- fate-polyacrylamide gel electrophoresis by matching the molecular weight with peaks on WCX2 chips and was identified as Serum Amyloid A protein (SAA) by MALDI/MS-MS and database searching. It was further validated in the same serum samples by immunoprecipitation with commercial SAA antibody. To confirm the SAA differential expression in lung cancer patients, the same set of serum samples was measured by ELISA assay. The result showed that at the cutoff point 0.446(OD value)on the Receiver Operating Characteristic (ROC) curve, SAA could better discriminate lung cancer from healthy indi- viduals with sensitivity of 84.1% and specificity of 80%. These findings demonstrated that SAA could be characterized as a biomarker related to pathological stages of lung cancer.     

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na . Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na . These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in △slr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress re-sponse by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.     

Purification and characterization of phenoloxidase from Octopus ocellatus

Phenoloxidase （PO） from ink sacs of Octopus ocellatus was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its biochemical and enzymatic properties by using L-dihydroxyphenylalanine （L-DOPA） as the specific substrate. It was found that prophenoloxidase from O. ocellatus was isolated as a heterodimeric protein of 153.8 kDa, and two subunits of 75.6 and 73.0 kDa were often detected in preparations after SDS activation. The PO-like activity showed optimal pH of 7.0, optimal temperature of 40℃, and an apparent Km value of 3.1 mM on L-DOPA, and 6.3 mM on catechol, respectively. The PO-like activity was extremely sensitive to 1-phenyl-2-thiourea and sodium suifite, and very sensitive to ascorbic acid, thiourea, citric acid, and benzoic acid. Together with its specific enzyme activity on catechol and L-DOPA, it can be concluded that the Octopus PO is most probably a typical o-diphenoloxidase. The PO-fike activity was also strongly inhibited by Cu^2＋, Zn^2＋, ethylenediaminetetraacetic acid and diethyldithiocarbamate （DETC）, and the DETC-inhibited PO-like activity could be perfectly restored by Cu^2＋. These results indicated that Octopus PO is most probably a copper-containing metalloenzyme. All these results implied that the PO from O. ocellatus has the properties of a catechol-type copper-containing o-diphenoloxidase which functions not only as a catalytic enzyme in melanin production in ink sacs but also as a humoral factor in host defense via melaninization as in other crustaceans.     

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase （asclepain c-II） has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. （Asclepiadaceae）. Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography （FPLC）. Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantrollne, phenyimethanesulfonyl fluoride, and pepstatine. The N-terminal sequence （LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA） showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-L-Gln-p-nitrophenyl ester as substrate： Km = 0.1634 mM, kcat = 121.48 s^-1, and kcat/Km = 7.4 ×10^5 s^-1/mM.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

光呼吸途径中两种转氨酶的研究进展

植物丝氨酸：乙醛酸氨基转移酶（SGAT）与谷氨酸：乙醛酸氨基转移酶（GGAT）主要催乙醛酸的转氨反应,是光呼吸途径中的两种关键酶。此两种酶大都为二聚体,在高等植物体内主要位于过氧化物酶体内,而在真核藻类植物体内则位于线粒体内,对植物的生长发育与抗逆性具有重要影响。本文对SGAT与GGAT在植物光合作用、氨基酸代谢和抗逆性等方面的研究进展进行了综述,以期对SGAT与GGAT的研究有所帮助。     

Serine:glyoxylate aminotransferase from the seedlings of rye (Secale cereale L.)

Serine:glyoxylate aminotransferase (EC 2.6.1.45) from green parts of 7-day-old rye seedlings was purified 600-fold. Specific activity of the purified enzyme against L-serine and glyoxylate as substrates was 53.2 mumol/mg protein per minute at 30 degrees C. The enzyme activity with L-alanine or L-asparagine and glyoxylate, or with L-asparagine and hydroxypyruvate was 20% that with L-serine and glyoxylate as the amino group acceptor, whereas with L-alanine or glycine and hydroxypyruvate it was 10% of that value. The reaction rate with pyruvate and L-asparagine, glycine or L-serine was very low. The enzyme was stabilized by the presence of sucrose, pyridoxal phosphate and 2-mercaptoethanol. Molecular sieving of the native enzyme on Sephacryl S-300 gel gave Mr values of 91,200 and 85,000, whereas the molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 43,000, indicating the dimeric structure of the enzyme.     

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.     

On the possibility of involvement of glutamate:glyoxylate and serine:glyoxylate aminotransferases from rye (Secale cereale L.) seedlings in the metabolism of tetrapyrrole compounds.

The activity of highly purified L-serine:glyoxylate aminotransferase (SGAT, EC 2.6.1.45) from rye seedlings was inhibited competitively by 5-aminolevulinate (ALA, Ki = 5 mM) SGAT was activated by hematin. Protoporphyrin IX and hematin inhibited irreversibly the activity of highly purified glutamate:glyoxylate aminotransferase (GGAT, EC 2.6.1.2) from rye seedlings. SGAT was found to catalyse transamination between ALA and hydroxypyruvate, whereas GGAT that between ALA and 2-oxoglutarate or pyruvate. It is suggested that SGAT is involved in the process of degradation of the excess ALA which has not been incorporated into porphyrin compounds.     

Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates.

Alanine: glyoxylate aminotransferase (EC 2.6.1.44), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized. The enzyme had Mr about 80 000, with two identical subunits. It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor. The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively. The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively). The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells. When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period. Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The primary deuterium isotope effect using L-serine 2-D is one on (V/K)serine and V in the steady state. Pre-steady-state experiments also indicate that there is no primary deuterium isotope effect with L-serine 2-D. The results suggest there is no rate limitation by abstraction of the alpha proton of L-serine in the SGAT reaction. In the steady-state a solvent deuterium isotope effect of about 2 was measured on (V/K)L-serine and (V/K)ketomalonate and about 5.5 on V. Similar solvent isotope effects were observed in the pre-steady-state for the natural substrates and the alternative substrate ketomalonate. In the pre-steady-state, no reaction intermediates typical of PLP enzymes were observed with the substrates L-serine, glyoxylate, and hydroxypyruvate. The data suggest that breakdown and formation of the ketimine intermediate is the primary rate-limiting step with the natural substrates. In contrast, using the alternative substrate ketomalonate, pre-steady-state experiments display the transient formation of a 490 nm absorbing species typical of a quinonoid intermediate. The solvent isotope effect results also suggest that with ketomalonate as substrate protonation at C(alpha) is the slowest step in the SGAT reaction. This is the first report of a rate-limiting protonation of a quinonoid at C(alpha) of the external Schiff base in an aminotransferase reaction.     

Glutamate and serine as competing donors for amination of glyoxylate in leaf peroxisomes

When provided with glycollate, peroxisomal extracts of leaves of spinach beet (Beta vulgaris L. cv.) converted L-serine and L-glutamate to hydroxypyruvate and 2-oxoglutarate respectively. When approximately saturating concentrations of each of these amino acids were incubated separately with glycollate, the utilization of serine was greater than that of glutamate. The utilization of glutamate was substantially reduced by the presence of relatively low concentrations of serine in the reaction mixture, whereas even high concentrations of glutamate caused only small reductions in serine utilization. Over the entire range of concentrations of amino acids examined, serine was invariably the preferred amino-group donor, but this preference was abolished at higher concentrations of glyoxylate. Serine not only competed favourably for glyoxylate but also inhibited L-glutamate: glyoxylate aminotransferase (GGAT), the degree of inhibition depending upon the glyoxylate concentration. Studies of L-serine: glyoxylate aminotransferase (SGAT) and GGAT in partially purified extracts from spinach-beet leaves confirmed that serine competitively inhibited GGAT but glutamate did not affect SGAT. Both enzymes were inhibited by high glyoxylate concentrations, the inhibition being relieved by suitably high concentrations of the appropriate amino acid. It is concluded that at the low glyoxylate concentrations likely to occur in vivo, the preferential utilization of serine would ensure flux through the glycollate pathway to glycerate, but at higher concentrations of glyoxylate, both enzymes could be fully active in glyoxylate amination.Abbreviations SGAT L-serine: glyoxylate aminotransferase - GGAT L-glutamate: glyoxylate aminotransferase     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.