Reduction of Methionine Sulfoxide by Plants

A relaxed (rel) mutant was found among thirty spontaneous thiopeptin-resistant isolates of Streptomyces antibioticus strain 3720, an actinomycin-producing strain, which showed severely reduced ability to accumulate ppGpp during a nutritional shift-down. The pool size of GTP decreased markedly in the parental strain, but to a lesser extent in the rel mutant. The rel mutant did not show the induction of an enzyme, phenoxazinone synthase, which is involved in the biosynthesis of actinomycin. No negative effect of the rel mutation was observed on a constitutive enzyme, kynurenine formamidase, which also plays a role in actinomycin synthesis. The mutant also failed to produce melanin, but still retained the ability to form aerial mycelium and spores, although the onset of the formation of aerial mycelium was markedly delayed. Neither the phenoxazinone synthase activity nor the kynurenine formamidase activity was affected by ppGpp in vitro. It is suggested tha the stringent response (ppGpp) may be generally essential for the induction of enzymes involved in secondary metabolism.     

Development and Characterization of a Monoclonal Antibody to Phytosiderophores

Mugineic acid-family phytosiderophores (MAs) are low molecularweight chelators that are secreted by graminaceous plants, formcomplexes with soil Fe(III) and are essential for plant growth.Methods to detect MAs which include HPLC and radio-immunoassaywith polyclonal antibody require sophisticated equipment orradio-labelled MAs which are difficult to synthesize. Our objectivewas to develop a detection and quantitation system for MAs basedon monoclonal antibody specificity and technology. A monoclonalantibody was produced which reacts with nicotianamine (NA),deoxymugineic acid (DMA), mugineic acid (MA) and epi-hydroxymugineicacid (epi-HMA) in a competitive ELISA. Azetidine-2-carboxylicacid (A-2-C) was not reactive while N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid (A-2-C dimer) was partially reactive.The range of detection using the competitive ELISA is from 2x 10^–6 to 2 x 10^–7 M MAs. Besides detection andquantification of MAs, the potential uses for the monoclonalantibody are numerous and include affinity chromatography andimmunocytochemistry. (Received September 26, 1991; Accepted December 16, 1991)     

Methionine recycling as a target for antiprotozoal drug development

The development of new and effective ontiprotozool drugs has been difficult because of the close metabolic relationship between protozoa and mammalian cells. In this article, Michael Riscoe, Al Ferro and john Fitchen present their hypothesis for chemotherapeutic exploitation of methylthioribose (MTR) kinase, an enzyme critical to methionine salvage in certain protozoa. They propose that analogues of MTR if properly designed, would be converted to toxic products in organisms that contain MTR kinase but not in mammalian cells, which lack this enzyme.     

Methionine acts as a "magnet" in photoaffinity crosslinking experiments

Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-at-a-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the "Magnet Effect" of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.     

The N-Terminal Methionine of Cellular Proteins as a Degradation Signal

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Methionine analogues as inhibitors of methionyl-tRNA synthetase

A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, -methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with K_i = 19 μM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.     

A Mediator of Rho-dependent Invasion Moonlights as a Methionine Salvage Enzyme

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.Cutaneous malignant melanoma has doubled in incidence over the past 30 years. Stages involved in progression of melanocytes to melanoma based on clinical and histopathological features include nontumorigenic nevi, dysplastic or atypical nevi, primary radial growth phase and vertical growth phase melanoma, and metastatic melanoma (1). Metastatic melanomas are often resistant to treatment; therefore therapeutic strategies require a more complete understanding of molecular determinants of this disease, particularly those involved in the invasive phenotype (2).Rho GTPases control a wide range of cellular responses including cell movement, morphogenesis, and coordinated migration (3). These pathways are implicated in malignant cell transformation and metastasis based on in vitro evidence showing tumorigenic and invasive responses to enhanced signaling in cell lines. Studies have demonstrated that overexpression of RhoC enhances invasion and metastasis in mouse xenografts of human melanoma and lung cancer cell lines (4, 5). In addition, some human tumors show elevated expression of Rho GTPases and exchange factors and/or reduced expression of GTPase-activating factors (6–8). Signaling through RhoA promotes actin polymerization and stress fiber formation, providing cells with contractile force needed for cell movement. Rho-GTP interacts with various effectors, including Rho-activated kinase, which promotes actin-myosin assembly via phosphorylation of myosin light chain phosphatase (9), or diaphanous-related formins, which nucleate actin filaments and stabilize microtubules (10, 11). Studies of cultured melanoma cells have revealed an “amoeboid” invasion mechanism involving RhoA-dependent Rho-activated kinase activation and inactivation of Rac (12, 13).RhoA also controls the formation and turnover of focal adhesion contacts, which mediate interactions between extracellular matrix and the actin cytoskeleton (14, 15). Signaling involves activation of Src and focal adhesion kinase (FAK)¹ and subsequent tyrosine phosphorylation of proteins recruited to integrin receptor complexes (16). Embryonic cells from FAK^−/− mice lose motility and cannot be rescued with FAK harboring a Y397F autophosphorylation site mutation not because they fail to form focal adhesions but because they are unable to disassemble focal adhesions (17). Thus, Rho controls cell movement by modulating the turnover of focal adhesion complexes via FAK. However, the mechanisms by which Rho GTPases control FAK are incompletely understood.In this study, we report that RhoA was constitutively activated in melanoma cells in a stage-specific pattern with elevated activity in cells from metastatic tumors. We present a functional proteomics screen for molecular targets of RhoA from which we identified a previously uncharacterized human protein induced in response to constitutive RhoA activation. This protein promoted Rho-dependent cell invasion and cell motility and provided a novel link for regulation of FAK tyrosine phosphorylation by RhoA. Thus, we refer to it as “mediator of Rho-dependent invasion (MRDI).” Although human MRDI has not been studied previously, it shows close sequence similarity to a methylthioribose-1-phosphate isomerase, which functions in methionine salvage pathways characterized in bacteria and yeast. We demonstrated that MRDI indeed has methylthioribose-1-phosphate isomerase activity and is required for methionine salvage in human cells. We further demonstrated that the catalytic activity of MRDI is independent of its role in cell invasion. Thus, MRDI is a dual function protein with promiscuous roles both as a metabolic enzyme and as an effector of signaling and cancer cell invasion.     

Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

贵州毕节喀斯特地区优势植物种子萌发特性初步研究

对采自贵州毕节地区的11种植物的种子萌发特性进行了初步研究,结果表明：①盐肤木、火棘、化香、云贵金丝桃与白栎种子在4周之内能够萌发;除云贵鹅耳枥胚坏死之外（萌发实验前后对种子进行解剖）,其他5种植物的种子都未萌发,处于不同的休眠状态。②盐肤木、化香、云贵金丝桃的种子光照时的萌发率远高于黑暗时的萌发率,具有显著差异,尤其是云贵金丝桃,因此3种植物种子均属于喜光性种子;而火棘与白栎种子有无光照都可以萌发,而且萌发率没显著差异,因此属于光不敏感或光中性种子。③盐肤木、云贵金丝桃的种子在30℃较高温条件下萌发最好;白栎、火棘种子在15℃、20℃低温条件下萌发更好;化香种子萌发温度既不能低于20℃也不能高于25℃。④刺异叶花椒种子吸水率高达85%,胚包埋在胚乳之中非常微小、未分化,因此可以初步判定属于形态休眠或者形态生理休眠;而平枝荀子、西域旌节花、云南旌节花种子吸水率都在20%以上,胚长/种子长都超多1/2,并且胚已发育完全,应属于生理休眠;小果蔷薇种子吸水率约27%,胚长/种子长都达2/3,并且通过对种子的解剖发现胚还未发育,应属于形态生理休眠。     

洞庭湖四种优势湿地植物茎、叶通气组织的比较研究

对通气组织的解剖观察有助于了解湿地植物的生长、分布及对不同生境的适应。采用石蜡切片技术,在光学显微镜下对洞庭湖湿地沿水位高程梯度分布的4种优势植物——荻Miscanthus sacchariflorus、水蓼Polygonum hydropiper、红穗苔草Carex argyi、虉草Phalaris arundinacea的茎和叶解剖结构进行了比较研究。结果表明：茎通气组织的形成部位主要在皮层、维管束和髓腔,其中髓腔所占比例最大（〉77%）。茎通气组织大小为：水蓼（57.8%）〉红穗苔草（45.5%）≥虉草（41.7%）≥荻（37.8%）。4种湿地植物的叶均在叶肉组织和（或）维管束内形成通气组织,如荻、虉草的形成部位是维管束,水蓼的是叶肉组织,而红穗苔草在叶肉组织和维管束内均可以形成,但以叶肉组织中为主,占99%。红穗苔草叶通气组织最发达,为33.8%,其它3种植物相对不发达,仅为0.13%～1.68%。除虉草外,其它3种植物通气组织大小与其分布位置具有很好的一致性。可见,湿地植物通气组织与其分布有较好的相关性。     

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C²-Man-)Trp.     

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer's disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.     

Precursor of mitochondrial glutamic oxaloacetic transaminase isozyme exists as a dimer

Mitochondrial glutamic oxaloacetic transaminase is synthesized in the cytoplasm as a larger precursor and then imported into mitochondria in association with its processing to mature form. The precursor corresponded to about 0.05-0.1% of the total proteins synthesized on membrane-free polysomes. Gel permeation chromatography showed that the molecular weight of the precursor was about 100,000 daltons, which was slightly larger than that of mature enzyme (homodimer: 45,000 x 2). On the other hand, sodium dodecylsulfate treated precursor had the molecular size (about 50,000 daltons) slightly larger than that of the subunit of mature one by the gel permeation chromatography. These results suggest that the precursor of mitochondrial glutamic oxaloacetic transaminase exists as a dimer.     

Exogenous Methionine as a Nutrient Supplement for the Induction of Xylogenesis in Lettuce Pith Explants

The induction of xylogenesis in explants of lettuce pith parenchymawas greatly influenced by the presence of exogenous methionine(0·025 to 0·05 µM) in the culture medium.At the various concentrations of methionine tested, trachearyelement differentiation was stimulated in the majority of theexplants. Differentiation, however, was markedly depressed ina small number of explants grown under the same cultural conditionsin the presence of methionine. Cytodifferentiation in controlexplants, cultured on a similar medium lacking methionine, gaveconsistent tracheary cell counts with little variation. Thesedata are consistent with the hypothesis that theenhanced productionof ethylene, due to the presence of methionine as a substrate,plays a role in the initiation of xylem differentiation. Theconcentration of ethylene in the cultured tissue may be a criticalfactor in determining whether the hormone will stimulate orsuppress the initiation of cytodifferentiation. Some thick-walledand pitted cells were observed, and these may represent partially-differentiatedxylem elements.     

Regional Distribution of Methionine Adenosyltransferase in Rat Brain as Measured by a Rapid Radiochemical Method

Abstract: The distribution of methionine adenosyltransferase (MAT) in the CNS of the rat was studied by use of a rapid, sensitive and specific radiochemical method. The S -adenosyl-[methyl-¹⁴C] l -methionine ([¹⁴C]SAM) generated by adenosyl transfer from ATP to [methyl-¹⁴C] l -methionine is quantitated by use of a SAM-consuming transmethylation reaction. Catechol O -methyltransferase (COMT), prepared from rat liver, transfers the methyl-¹⁴C group of SAM to 3,4-dihydroxybenzoic acid. The ¹⁴C-labelled methylation products, vanillic acid and isovanillic acid, are separated from unreacted methionine by solvent extraction and quantitated by liquid scintillation counting. Compared to other methods of MAT determination, which include separation of generated SAM from methionine by ion-exchange chromatography, the assay described exhibited the same high degree of specificity and sensitivity but proved to be less time consuming. MAT activity was found to be uniformly distributed between various brain regions and the pituitary gland of adult male rats. In the pineal gland the enzyme activity is about tenfold higher.