Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli

A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite. N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2. The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman. In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside. The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein. The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence. The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5. The enzyme had a temperature optimum at 40 degrees C. The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.     

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.     

The two main endoproteases present in dark-induced senescent wheat leaves are distinct subtilisin-like proteases

We have previously reported the occurrence of two serine endoproteases (referred to as P1 and P2) in dark-induced senescent wheat (Triticum aestivum L.) leaves. P1 enzyme was already purified and identified as a subtilisin-like serine endoprotease (Roberts et al. in Physiol Plant 118:483–490, 2003). In this paper, we demonstrate by Western blot analysis of extracts obtained from dark-induced senescent leaves that an antiserum raised against P1 was able to recognise a second protein band of 78 kDa which corresponded to P2 activity. This result suggested that both enzymes must be structurally related. Therefore, we purified and characterised P2 activity. According to its biochemical and physical properties (inhibition by chymostatin and PMSF, broad pH range of activity, thermostability and ability to hydrolyse Suc-AAPF-pNA) P2 was classified as a serine protease with chymotrypsin-like activity. In addition, P2 was identified by mass spectrometry as a subtilisin-like protease distinct from P1. Western blot analysis demonstrated that P1 appeared in extracts from non-detached dark-induced senescent leaves but was undetectable in leaves senescing after nitrogen (N) deprivation. In contrast, P2 was already present in non-senescent leaves and showed increased levels in leaves senescing after N starvation or incubation in darkness. P1 signal was detected at late stages of ethephon or methyl jasmonate-induced senescence but was undetectable in senescent leaves from plants treated with abscisic acid. None of the three hormones have any effect on P2 protein levels. These results indicate that despite their biochemical and structural similarities, both enzymes are probably involved in different physiological roles.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit M(r) 22,000 having an optimum catalytic activity at pH 6.5-7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for K(m)(GST) and K(m)(CDNB) of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased temperatures, but both enzymes needed 10 min incubation at 60 degrees C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins.     

Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence

The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea (Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-utilization in the peroxisomal metabolism.     

Natural Senescence of Pea Leaves (An Activated Oxygen-Mediated Function for Peroxisomes)

We studied the activated oxygen metabolism of peroxisomes in naturally and dark-induced senescent leaves of pea (Pisum sativum L.). Peroxisomes were purified from three different types of senescent leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. The activities of the O2-- and H2O2-producing enzymes were enhanced by natural senescence. Senescence also produced an increase in the generation of active oxygen species (O2- and H2O2) in leaf peroxisomes and in the activities of two glyoxylate-cycle marker enzymes. A new fraction of peroxisomes was detected at an advanced stage of dark-induced senescence. Electron microscopy revealed that this new peroxisomal fraction varied in size and electron density. During senescence, the constitutive Mn-superoxide dismutase (SOD) activity of peroxisomes increased and two new CuZn-SODs were induced, one of which cross-reacted with an antibody against glyoxysomal CuZn- SOD. This fact and the presence of glyoxylate-cycle enzymes support the idea that foliar senescence is associated with the transition of peroxisomes into glyoxysomes. Our results indicate that natural senescence causes the same changes in peroxisome-activated oxygen metabolism as dark-induced senescence, and reinforce the hypothesis of an effective role of peroxisomes and their activated oxygen metabolism in this stage of the life cycle.     

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.     

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.     

Cloning and characterization of a glutathione S-transferase induced by a herbicide safener in barley (Hordeum vulgare)

The effect of the herbicide safener mefenpyr-diethyl on glutathione S -transferase (GST, EC 2.5.1.18) activities of dark-grown barley ( Hordeum vulgare cv. Alexis) was examined. Mefenpyr-diethyl treatment increased the GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) and the herbicide fenoxaprop as substrates. Glutathione (GSH) peroxidase activity was markedly increased. GST subunits were analysed by high performance liquid chromatography (HPLC). The quantities of two major subunits were increased by the safener treatment, while the quantities of two other major subunits remained constant. A cDNA encoding the most abundant inducible GST (HvGST6) was cloned and expressed in E. coli . The purified enzyme exhibited a low activity with herbicides as substrates. By contrast, it exhibited a strong GSH peroxidase activity.     

Purification and characterization of a novel sigma-class glutathione S-transferase of the fall webworm, Hyphantria cunea

Abstract:  An enzyme that possesses glutathione S -transferase (GST) activity was found in the fall webworm, Hyphantria cunea . The enzyme was purified to homogeneity for the first time by ammonium sulphate fractionation and affinity chromatography. The N-terminal sequence of the purified protein was similar to those of Sigma-class GSTs. The purified GST retained more than 75% of its original GST activity after incubation at pH 5–8. Incubation for 30 min at temperatures below 50°C scarcely affected the activity. The enzyme was able to catalyse the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation.     

Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.     

N-Methyltransferase involved in gramine biosynthesis in barley: cloning and characterization

The indole alkaloid gramine occurs in leaves of certain barley (Hordeum vulgare L.) cultivars but not in others. A gene sequence in barley that earlier was characterized as a jasmonate-induced O-methyltransferase (MT) (EC 2.1.1.6, GenBank accession U54767) was here found to be absent in some barley cultivars and breeding lines that all lacked gramine. The cDNA was cloned and expressed in Escherichia coli and the recombinant protein purified. The purified recombinant protein methylated two substrates in the pathway to gramine: 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) at a high rate, with Km-values of 77 microM and 184 microM, respectively. In contrast, the protein did not exhibit any detectable methylation with the earlier suggested substrate for O-methylation, caffeic acid. A number of cultivars and breeding lines of barley were analyzed for presence of the U54767 gene sequence and MT protein and the enzyme activity in vitro with MAMI or caffeic acid as substrates. The results showed a clear relationship between the presence of the MT gene, the MT protein and N-methyltransferase activity, and confirmed the identification of the gene as coding for an N-methyltransferase (NMT, EC 2.1.1) and being involved in gramine biosynthesis.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

水稻施用硅肥对稻纵卷叶螟幼虫保护酶和解毒酶活性的影响

【目的】研究施硅水稻对稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)幼虫保护酶(过氧化氢酶CAT、过氧化物酶POD、超氧化物歧化酶SOD)和解毒酶(谷胱甘肽-S-转移酶GST、乙酰胆碱酯酶ACh E)活性的影响,为探明硅增强水稻抗稻纵卷叶螟的机理和稻纵卷叶螟的综合治理提供依据。【方法】采用感虫水稻品种TN1,设置2种施硅水平,即0.32 g Si/kg土壤(Si+)和不施硅(Si-),测定取食水稻24、48、72、96 h后3龄幼虫体内CAT、POD、SOD、GST、ACh E活性的动态变化。【结果】幼虫取食Si+水稻前期(24~48 h),CAT、POD、SOD活性呈上升趋势,并在48 h时达到最高值;取食后期(48~96 h),CAT、POD、SOD活性下降并在96 h时显著低于Si-处理。在Si-处理中,幼虫CAT和POD活性"先降低、再升高",SOD活性呈一直增大的趋势。取食Si+水稻幼虫GST活性始终显著高于Si-处理,而ACh E活性均低于Si-水稻,并在72 h和96 h时显著低于Si-水稻。处理间幼虫存活率存在显著差异,Si+水稻上1龄和3龄幼虫的存活率显著低于Si-水稻。这些结果说明,硅处理可能使稻纵卷叶螟幼虫产生应激反应引起保护酶活性在短时间内增大,但取食一段时间后保护酶活性下降。ACh E活性受到抑制,会引起昆虫神经传导的异常反应,造成昆虫死亡。【结论】硅可能通过参与植物的生理代谢过程,诱导植物增加次生代谢物的合成和积累,引起稻纵卷叶螟保护酶和解毒酶的活性变化,可能会影响稻纵卷叶螟的存活。     

Chloroplast pigments,proteins, lipid peroxidation and activities of antioxidative enzymes during maturation and senescence of leaves and reproductive organs of Cajanus cajan L.

A comparative investigation was undertaken with pigeon pea leaves and attached flower buds/flowers/pods during their developmental stages including senescence in a natural system in experimental plots. Alterations in chloroplast pigments, total soluble proteins, lipid peroxidation, malondialdehyde (MDA) content and activities of guaiacol peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) were studied at 5-day interval from initial to 40-day stage. Chloroplast pigments and proteins of leaves increased upto 15 and 20-day stages respectively followed by a steady decline. Reproductive parts, however, exhibited rise in chloroplast pigments upto 25-day and protein till last stage as developing pods gain the amount from the senescing leaves which are nearest to them. Senescing leaves show very high POD activity than the developing and senescing pods and POD appears to be associated with chlorophyll degradation. Considerably higher activity and amount of LOX and MDA respectively have been noticed in senescing leaves than in flowers and pods. Increase in SOD activity during early stage of leaf growth and maturation indicates protective role that declined at senescent stages. Pods are unique in having very high SOD activity, only last stage of senescence does show a decline.