Binding Sites of the Polyamines Putrescine,Cadaverine, Spermidine and Spermine on A- and B-DNA Located by Simulated Annealing

Abstract

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na⁺ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.     

外源精胺、亚精胺作用下离体小麦叶片-老化过程中蛋白水解酶活性和内源精胺、亚精胺含量的变化(简报)1

外源精胺、亚精胺明显抑制离体小麦叶片老化过程中蛋白水解酶活性上升;小麦叶片老化期间内源精胺、亚精胺含量逐渐下降,与蛋白水解酶活性升高对应。     

Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells.     

紫杉醇生物合成相关酶基因的克隆与表达

以牛儿基牛儿基二磷酸为前体的紫杉醇生物合成大约有20步酶促反应,其反应过程已基本阐明,近一半的相关酶基因已得到克隆与表达。综述了编码参与紫杉醇生物合成的紫杉二烯合酶、紫杉二烯5α羟化酶、紫杉烷10β羟化酶、紫杉烷13α羟化酶、紫杉二烯5α醇O乙酰基转移酶、紫杉烷2α苯甲酰转移酶、去乙酰基巴卡亭Ⅲ10βO乙酰转移酶、3氨基3苯基丙酰转移酶和3N去苯甲酰2脱氧紫杉醇苯甲酰转移酶等9个酶基因的克隆和表达方面的研究情况,并指出随着紫杉醇生物合成的分子生物学研究的不断深入,利用分子生物技术大规模生产紫杉醇将为期不远 。     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic beta-(1,2)-Glucan Biosynthesis

The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.     

Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O₂ uptake rates, P700 photooxidation and the accumulation of superoxide anions (O₂ ⁻) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O₂ ⁻ generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O₂ ⁻ generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.     

Duplication and Diversification of the Spermidine/Spermine N1-acetyltransferase 1 Genes in Zebrafish

Spermidine/spermine N¹-acetyltransferase 1 (Ssat1) is a key enzyme in the polyamine interconversion pathway, which maintains polyamine homeostasis. In addition, mammalian Ssat1 is also involved in many physiological and pathological events such as hypoxia, cell migration, and carcinogenesis. Using cross-genomic bioinformatic analysis in 10 deuterostomes, we found that ssat1 only exists in vertebrates. Comparing with mammalian, zebrafish, an evolutionarily distant vertebrate, contains 3 homologous ssat1 genes, named ssat1a, ssat1b, and ssat1c. All zebrafish homologues could be transcribed and produce active enzymes. Despite the long history since their evolutionary diversification, some features of human SSAT1 are conserved and subfunctionalized in the zebrafish family of Ssat1 proteins. The polyamine-dependent protein synthesis was only found in Ssat1b and Ssat1c, not in Ssat1a. Further study indicated that both 5′ and 3′ sequences of ssat1b mediate such kind of translational regulation inside the open reading frame (ORF). The polyamine-dependent protein stabilization was only observed in Ssat1b. The last 70 residues of Ssat1b were crucial for its rapid degradation and polyamine-induced stabilization. It is worth noting that only Ssat1b and Ssat1c, but not the polyamine-insensitive Ssat1a, were able to interact with integrin α9 and Hif-1α. Thus, Ssat1b and Ssat1c might not only be a polyamine metabolic enzyme but also simultaneously respond to polyamine levels and engage in cross-talk with other signaling pathways. Our data revealed some correlations between the sequences and functions of the zebrafish family of Ssat1 proteins, which may provide valuable information for studies of their translational regulatory mechanism, protein stability, and physiological functions.     

A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.     

大麦幼苗多胺合成比脯氨酸合成对盐胁迫更敏感

NaCl 2 0 0mmol/L处理结合14 C Glu叶面饲喂 6天龄大麦幼苗 ,结果证明盐胁迫下Pro主要积累在叶片中 ,在根系中PA的积累占优势。PA合成途径对盐胁迫的响应早于Pro。盐处理 8h以后PA与Pro的合成竞争共同前体Arg。盐胁迫激活了Pro两条合成途径 ,胁迫 8h以前Pro积累主要受Glu途径控制 ,随后Orn途径对Pro积累的贡献占主导地位。盐胁迫促进了PA合成的Arg途径 ,对Orn途径没有影响     

渗透胁迫对小麦胚芽鞘内多胺的种类、形态和含量的影响

用高压液相色谱法研究了豫麦18（抗旱性较强）和扬麦9号（抗旱性较弱）小麦胚芽鞘中三种不同形态的多胺（polyamine,PA）：游离态多胺（PA）、高氯酸可溶性结合态多胺（ps结合态PA）和高氯酸不溶性结合态多胺（PIS结合态PA）与渗透胁迫的关系。结果发现：渗透胁迫2d,豫麦18胚芽鞘中的游离态Spd和游离态Spm的含量明显上升,而扬麦9号的游离态Put的上升明显。S-腺苷蛋氨酸脱羧酶（S—AMDC）的抑制剂——甲基乙二醛-双（鸟嘌呤腙）（MGBG）处理豫麦18,明显抑制了渗透胁迫诱导的游离态Spd和游离态Spm的增加,并且加重了渗透胁迫伤害,外源Spd处理扬麦9号明显促进了渗透胁迫诱导的游离态Spd和游离态Spm的增加,并且减缓了渗透胁迫的伤害。渗透胁迫下,豫麦18胚芽鞘中的PS结合态PA和PIS结合态PA的上升幅度都明显大于扬麦9号。菲咯啉（o—Phen）抑制渗透胁迫下PIS结合态PA的合成并加重了渗透胁迫对胚芽鞘的伤害。这些结果表明：小麦胚芽鞘中的游离态Spd、游离态Spm、PS结合态PA和PIS结合态PA的升高有利于增强渗透胁迫抗性。     

Streptomycin Resistance (rpsL) Produces an Absolute Requirement for Polyamines for Growth of an Escherichia coli Strain Unable to Synthesize Putrescine and Spermidine [Δ(speA-speB) ΔspecC]

The presence of certain rpsL (strA) mutations in a strain of Escherichia coli that cannot synthesize putrescine or spermidine because of deletions in ornithine decarboxylase, arginine decarboxylase, and agmatine ureohydrolase, converts a partial requirement for polyamines for growth into an absolute requirement.     

Spermine Promotes Acclimation to Osmotic Stress by Modifying Antioxidant, Abscisic Acid, and Jasmonic Acid Signals in Soybean

The possible involvement of spermine (Spm) in the acclimation of soybean to osmotic stress was investigated by determining the changes in photosynthetic pigments, antioxidants, and plant hormone levels in response to applied Spm. Plants were exposed to 9 % PEG-induced osmotic stress with or without 0.4 μM Spm. Osmotic stress reduced the relative water content, chlorophyll a, chlorophyll b, carotenoid, and protein contents in leaves, and these detrimental effects were alleviated by treatment with Spm. Moreover, the significant increase in the content of abscisic acid and decrease in that of jasmonic acid in plants subjected to osmotic stress was attenuated by treatment with Spm. Osmotic stress caused a significant increase in lipid peroxidation when compared to controls, and that was accompanied by a slight reduction in the level of antioxidants and reduced glutathione and in the activities of catalase, superoxide dismutase, peroxidase, and polyphenol oxidase. Spm treatment ameliorated these osmotic stress effects by reducing lipid peroxidation and increasing catalase, superoxide dismutase, peroxidase, and polyphenol oxidase activities. These results indicate that application of Spm could be exploited to alleviate a moderate level of osmotic stress through the regulation of stress-related components such as photosynthetic pigments, plant hormones, and antioxidants.     

外源腐胺对根际低氧胁迫下黄瓜幼苗多胺和抗氧化系统的影响

采用营养液栽培,研究了外源腐胺(Put)对根际低氧胁迫下黄瓜幼苗体内多胺含量和抗氧化系统的影响.结果显示,低氧胁迫显著刺激了黄瓜幼苗体内活性氧(ROS)和内源多胺含量的增加,提高了抗氧化酶活性;外源Put进一步提高了低氧胁迫下黄瓜幼苗体内多胺的含量和抗氧化酶活性,降低了ROS含量,从而缓解了低氧胁迫的伤害作用;Put合成抑制剂D-精氨酸(D-Arg)不仅显著抑制黄瓜幼苗体内多胺的合成,而且抑制抗氧化酶活性,同时ROS大量积累,进一步抑制黄瓜幼苗的生长;而外源Put可缓解D-Arg的抑制作用;Put转化抑制剂甲基乙二醛-双(脒基腙)(MGBG)和Put降解抑制剂氨基胍(AG)的混合施用造成游离态Put的过量积累,以及亚精胺(Spd)、精胺(Spm)含量和抗氧化酶活性的显著降低,造成ROS大量积累,进一步加重了低氧胁迫对植株的伤害.结果表明,低氧胁迫下外源Put可提高黄瓜幼苗体内游离态Put含量,促进游离态Put向Spd和Spm转化,Spd、Spm含量的增加以及(free-Spd free-Spm)/free-Put比值的升高有利于提高植株抗氧化酶活性,增强清除ROS的能力,降低膜脂过氧化的伤害,从而增强植株的低氧胁迫耐性.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic β-(1,2)-Glucan Biosynthesis

The cyclic β-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic β-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic β-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic β-(1,2)-glucan biosynthesis.     

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-¹⁴C]putrescine, the specific radioactivity (sra) of the newly formed [¹⁴C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-¹⁴C]methionine, the other polyamine precursor, significantly higher sra values for [¹⁴C]spermidine and [¹⁴C]spermine were recorded in the brains of the MSO-treated animals. [¹⁴C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-¹⁴C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-¹⁴C]-methionine. The results of these experiments show both significantly lower sra values for [¹⁴C]spermidine and [¹⁴C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.     

The Biosynthesis of Polyamines in the Brain of Audiogenic Seizure-Susceptible and -Resistant Deermice

Abstract: The biosynthesis of polyamines was investigated in the brains of the audiogenic seizure-susceptible (SS) mutant and the wild-type, seizure-resistant (SR) deermouse Peromyscus maniculatus bairdii. For this purpose a new, rapid, and economical high pressure liquid chromatography (HPLC) procedure for the quantitation of putrescine, spermidine, and spermine was developed. Benzoyl derivatives of the polyamines, prepared from a crude brain supernatant, were ether extracted and, following removal of the ether, were separated and quantitated by HPLC. The high sensitivity of the method allows quantitation of putrescine in 50 mg and of spermidine and spermine, in as little as 2-2.5 mg, of brain tissue. No differences were found in endogenous levels of the 3 polyamines in brains of SS vs SR deermice. Using [¹⁴C]putrescine as a polyamine precursor, we found the specific radioactivity of spermidine to be lower in the SS than in the SR brains following a 1 h intraventricular (i.vt.) pulse. No such differences were noted if [3,4-¹⁴C]methionine was used as the polyamine precursor. To test whether the flux of methionine through the transmethylation pathway was also different in SS and SR deermouse brain, we administered [1-¹⁴C]methionine (i.vt.) (1 h pulse). Even though the brains of SS animals contained higher methionine and lower S-adenosyl-l -methionine (AdoMet) levels than the SR brains, the specific radioactivities of methionine and AdoMet were, respectively, lower and higher in SS compared to SR brains. The latter results are in agreement with our previous findings of an accelerated utilization of AdoMet in brains of Swiss-Webster mice following administration of the chemical convulsant l -methionine-d,l-sulfoximine (MSO). Taken together, the data suggest that the SS condition, whether genetically determined (as in the SS deermouse) or chemically elicited (as after MSO), correlates positively with higher than normal rates of conversion of methionine to brain AdoMet and leads to an enhanced rate of utilization of AdoMet via the transmethylation pathway.     

Polyamine Metabolism in the Roots of Phaseolus vulgaris. Interaction of the Inhibitors of Polyamine Biosynthesis with Putrescine in Growth and Polyamine Biosynthesis

The effects of the inhibitors of polyamine biosynthesis, canavanineand -methyl ornithine on growth, the activities of argininedecarboxylase (EC 4.1.1.19 [EC] ) and ornithine decarboxylase (EC4.1.1.17 [EC] ) and on polyamine content were examined in two differentgrowth regions of Phaseolus vulgaris L. cv. Taylor's Horticulturalroots. Separately, in the same manner, in the same bean rootsystem exogenous putrescine effect and the interaction of canavaninewith putrescine were determined. The arginine and ornithine decarboxylase activities found inroot apex were high where cell division activity was highest.Polyamine (putrescine and spermine) content did not correlatewith these activities, but polyamine level was high in the rootbase where cell elongation is the main process. The arginineanalogue, canavanine, inhibited arginine decayboxylase activityand polymine liters. Putrescine partially reversed the canavanineinhibition of root growth as well as arginine decarboxylaseactivity and polyamine content. Similarly -methyl ornithineslightly inhibited the root length and ornithine decarboxylaseactivity in the root apex. Besides, exogenous putrescine didnot effect significantly the endogenous polyamine titers. Theseresults reinforce the growing connection between polyaminesand the rates of cell devision in the roots of bean plants.Separately, arginine decarboxylase is the main enzyme in thebean roots. (Received November 10, 1986; Accepted March 3, 1987)