Demonstration and solubilization of S-adenosylmethionine: γ-tocopherol methyltransferase from Capsicum chromoplasts

Through the use of Capsicum chromoplast membranes, we report for the first time the direct methylation of -tocopherol into -tocopherol in the presence of S-adenosylmethionine. Furthermore the S-adenosylmethionine: -tocopherol methyltransferase activity has been solubilized. On a protein basis, the activity recovered in the soluble preparation was higher than that bound to the membranes.     

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T₂ seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.     

Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase

Alpha-tocopherol (vitamin E) is synthesized from gamma-tocopherol in chloroplasts by gamma-tocopherol methyltransferase (gamma-TMT; VTE4). Leaves of many plant species including Arabidopsis contain high levels of alpha-tocopherol, but are low in gamma-tocopherol. To unravel the function of different forms of tocopherol in plants, an Arabidopsis plant (vte4-1) carrying a functional null mutation in the gene gamma-TMT was isolated by screening a mutant population via thin-layer chromatography. A second mutant allele (vte4-2) carrying a T-DNA insertion in the coding sequence of gamma-TMT was identified in a T-DNA tagged mutant population. In vte4-1 and vte4-2 leaves, high levels of gamma-tocopherol accumulated, whereas alpha-tocopherol was absent indicating that, presumably, these two mutants represents null alleles. Over-expression of the gamma-TMT cDNA in vte4-1 restored wild-type tocopherol composition. Mutant plants were very similar to wild type. During oxidative stress (high light, high temperature, cold treatment) the amounts of alpha-tocopherol and gamma-tocopherol increased in wild type, and gamma-tocopherol in vte4-1. However, chlorophyll content and photosynthetic quantum yield were very similar in wild type and vte4-1, suggesting that alpha-tocopherol can be replaced by gamma-tocopherol in vte4-1 to protect the photosynthetic apparatus against oxidative stress. Fatty acid and lipid composition were very similar in WT, vte4-1 and vte1, an Arabidopsis mutant previously isolated which is completely devoid of tocopherol. Therefore, a shift in tocopherol composition or the absence of tocopherol has no major impact on the amounts of specific fatty acids or on lipid hydrolysis.     

Molecular cloning and characterization of gene coding for γ-tocopherol methyltransferase from lettuce (Lactuca sativa)

γ-tocopherol methyltransferase is an important rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding γ-tocopherol methyltransferase (designated as LsTMT) was cloned from Lactuca sativa for the first time by rapid amplification of cDNA ends and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTMT was 1131 bp, with an open reading frame of 897 bp encoding a γ-tocopherol methyltransferase protein of 298 amino acids, with a calculated molecular mass of 33.06 kDa and an isoelectric point of 5.86. Comparative analysis revealed that LsTMT has a close similarity with γ-TMTs from other plant species. Bioinformatic analysis indicated that LsTMT shares a common evolutionary origin based on sequence similarity and has the closest relationship to γ-TMT from the sunflower, Helianthus annuus. Based on quantitative RT-PCR analysis, we found that expression of LsTMT is induced and strengthened by oxidative stresses such as strong light and drought. The cloning and characterization of LsTMT will be helpful to further understanding its role in the tocopherol biosynthesis pathway. We consider it to be a candidate gene for metabolic engineering of vitamin E in vegetable crops.     

Participation of β-carotene in reactivation of PSI of heptane-extracted spinach chloroplasts

A carotenoid requirement for photosystem I activity in spinach chloroplasts using extraction-reconstitution technique has been investigated. The transfer of electron from N,N,N,N-tetramethyl-p-phenylene diamine through the chloroplast photosystem to methyl viologen dye or to NADP⁺ was used as an assay of photosystem I activity. Extraction of lyophilized spinach chloroplasts with heptane at near 0°C removed almost all -carotene and reduced photochemical activities associated with photosystem I to a low level (about 15% of the original activity). Reconstitution of the extracted chloroplasts with -carotene completely restored photosystem I activity. The maximum rate of methyl viologen photoreduction in reconstituted chloroplasts occurred at an -carotene/chlorophyll molar ratio of 0.5. Cyclic phosphorylation mediated by phenazine methosulphate was partially restored. Xanthophylls (lutein, neoxanthin, violaxanthin), as components of chloroplast membranes, were not able to replace -carotene in reconstitution of chloroplasts and had essentially no effect on restoring photoreactions. On the basis of the P700/total chlorophyll ratio it can be assumed that extraction of lyophilized chloroplasts with heptane do not affect photosystem I reaction centre. Therefore it is possible that -carotene, removed during heptane extraction and belonging mainly to the antenna pigment pool of photosystem I, is effective in the restoration of photosystem I activity.Abbreviations chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - MV methyl viologen - PMS phenazine methosulphate - PQA plastoquinone A - PS I photosystem I - PS II photosystem II - TMPD N,N,N',N'-tetramethyl-p-phenylene diamine - Tricine N-tris(hydroxymethyl)methyglycine. D-1, D-10, D-50, D-144 represent chloroplast subfractions sedimented at 1000 × g, 10,000 g, 50,000 × g and 144,000 × g - s supernatant This paper is a partial fulfillment of the requirements for the Ph.D. degree of A.T. at Maria Curie-Skodowska University, Lublin.     

Overexpression of the maize γ-tocopherol methyltransferase gene (ZmTMT) increases α-tocopherol content in transgenic Arabidopsis and maize seeds

Transgenic Research - The vitamin E family includes tocopherols and tocotrienols, which are essential lipid-soluble antioxidants necessary for human and livestock health. The seeds of many plant...     

Nitration of γ-tocopherol in plant tissues

Nitration of γ-tocopherol has been suggested to be an important mechanism for the regulation and detoxification of reactive nitrogen oxide species in animal tissues. To investigate whether this reaction does also occur in plants, reversed phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) were used for analysis of 5-nitro-γ-tocopherol (5-NγT) in leaves and seeds. 5-nitro-γ-tocopherol (5-NγT) could be detected in an in vitro system where it was most likely generated by the reaction of γ-tocopherol with a nitric oxide radical. In vivo 5-NγT was identified in leaves of the Arabidopsis mutant line (vte4), which has insertion in the gene encoding γ-tocopherol methyltransferase and consequently lacks α-tocopherol and accumulates high levels of γ-tocopherol. Quantification of NO_x in leaves revealed that the vte4 mutant in comparison to wild type and the mutant vte1, which does not contain any tocopherol, has a reduced NO_x concentration. The level of 5-NγT in leaves of the vte4 mutant was shown to depend on the developmental stage and on the duration of light exposure. 5-NγT was also detectable in germinating seeds of Brassica napus, Nicotiana tabacum and Arabidopsis thaliana. These seeds have in common high γ-tocopherol contents. The rate of germination at two days after imbibition inversely correlated with the γ-tocopherol content of the seeds. The result suggests that γ-tocopherol or its respective derivative, 5-NγT, may prolong early development by reducing the level of NO_x.     

Polypeptide profiles of chlorophyll · protein complexes and thylakoid membranes of spinach chloroplasts

In addition to the major chlorophyll · protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll $\text{a}\text{b}$ · protein and from the reaction centre complex of Photosystem II.Data are presented on the polypeptide profiles of purified digitonin-subchloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll · protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.     

Some properties of A β-1,3-glucanase from rye

Molecular-sieve chromatography of an extract from ungerminated rye indicated the presence of enzymes which hydrolysed cellobiose, laminaribiose and the β-glucans cellodextrin, laminarin and barley β-glucan. A purified endo-β-1,3-glucanase was prepared from the extract by ammonium sulphate fractionation and molecular-sieve chromatography on Biogel P60. The substrate specificity and some properties of the enzyme are reported and the in vivo role of the enzyme is discussed.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Solubilization and some properties of γ-glutamyltransferase from human brain microvessels

Detergents Triton X-100, sodium deoxycholate, and octyl--D-glucopyranoside, and proteinase papain proved to be excellent agents solubilizing the -glutamyl-transferase (-GT) from human brain cortex microvessels. Ficin also solubilized -GT but to a lesser extent than papain. The relative molecular mass of the detergent-solubilized enzyme form was greater than 200,000 (in the presence of Triton X-100). The relative molecular mass of the proteinase-solubilized form was slightly greater than that of albumine. -GTs of microvessels from five human brain regions and from the choroid plexus were tested for their specificity toward acceptors. The best acceptors were found to be (in decreasing order of activity)l-cystine, glycylglycine,l-glutamine,l-methionine, andl-alanine. The findings suggest that the main features of -GT of the human blood-brain barrier are very similar to those of -GTs from other human tissues.     

Purification and properties of γ-glutamyl transferase from normal rat liver

γ-Glutamyl transferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) has been partially purified from both whole rat liver (600-fold) and from isolated biliary tract (1200-fold). The most highly purified fraction gave two protein bands on polyacrylamide gel electrophoresis, the major band alone having enzyme activity. The enzyme purified from biliary tract appears identical to that from whole liver preparation according to molecular weight, kinetic parameters and the effects of various inhibitors.Three liver cell-types; parenchymal, Kupffer and biliary tract were isolated by perfusion of the rat liver in situ with collagenase, followed by selective cell isolation. Approx. 80–90% of the total recovered enzyme activity was found in the biliary tract. Nearly 50% of the apparent enzyme activity in the parenchymal cell was attributable to a nonspecific hydrolase.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Rheological and structural properties of starches from γ-irradiated and stored potatoes

Starch was extracted from irradiated and stored potato tubers and the properties were compared to CIPC (chlorpropham) treated tubers. The granule properties and dynamic viscoelasticity in temperature ramp and frequency sweep modes were studied while heating the samples. Starch structural characteristics were investigated by high performance anion exchange chromatography (HPAEC) and Fourier transform infrared spectroscopy (FTIR). Gamma-irradiation of potato tubers at a dosage of 0.1 kGy induced some degradation of starch molecules, resulting in earlier swelling of starch granules, and greater extents of amylose and total carbohydrate leaching. The early swelling phenomenon was also enhanced with tuber storage time. The retrogradation rate and extent for a concentrated starch gel also increased with tuber storage time whereas γ-irradiation delayed the gel retrogradation. Sprout inhibiting methods could be selected based on the specific processing and texture requirements of the end products.     

Effects of reactive oxygen species on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis

The effects of reactive oxygen species (ROS) on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis were investigated. Addition of an organic carbon source to the medium resulted in increased mitochondrial activity, intracellular O₂ ^- concentration and α-tocopherol productivity in E. gracilis W₁₄ZUL (a chloroplast deficient mutant). α-Tocopherol productivity of the wild-type strain (with both mitochondria and chloroplast) was higher than that of the W₁₄ZUL strain. In the case of the wild strain, the O₂ ⁻ generated in chloroplasts was efficiently scavenged by the α-tocopherol synthesized inside the chloroplast. In photoheterotrophic culture (with an organic carbon source), there was a positive correlation between α-tocopherol production and O₂ ⁻ generation. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) resulted in increased O₂ ⁻ generation and α-tocopherol productivity. These results indicate that the ROS generated in mitochondria and chloroplasts play important roles in α-tocopherol production by E. gracilis. The presence of chloroplasts and generation of intracellular ROS are important for efficient production of α-tocopherol.     

Enzymic synthesis of γ-coniceine in Conium maculatum chloroplasts and mitochondria

Further studies of the transaminase responsible for the first committed step in alkaloid formation in Conium maculatum have shown the L-alanine: 5-ketooctanal transaminase to occur in both the mitochondria and chloroplast. Experiments suggest that these enzymes are the isoenzymes Transaminase A and B respectively previously isolated by the author. It is suggested that the chloroplast enzyme is normally responsible for alkaloid production.