台琼海桐蒴果皮油及籽油的主要挥发性化学成分

采用GC-MS技术,对台琼海桐蒴果皮的水蒸气蒸馏油样,以及蒴果籽的索氏提取油样的甲酯化样品进行分析。样品中各化学成分的相对含量用面积归一化法确定。在实验条件下,果皮油样共分离出24个峰,鉴定了17个,占总峰面积的94.70%。其主要成分为柠檬烯(24.27%)、γ-榄香烯(8.30%)、β-榄香烯(17.13%)、α-榄香烯(16.02%)、长叶龙脑(15.52%)和α-松油醇(2.17%),α-石竹烯(1.88%)。果籽油的甲酯化样品共分离出16个峰,鉴定了12个,占总峰面积的96.69%,其主要成分为棕榈油酸(34.83%)、反油酸(26.63%)、14-甲基-十五烷酸(17.08%),十八烷酸(1.88%),十四烷酸(1.61%)。分析结果说明台琼海桐蒴果皮富含挥发性芳香油,而蒴果籽油含有多种脂肪酸。     

Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding

Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.     

Determination of ribonucleoside triphosphates and deoxyribonucleoside triphosphates in Novikoff hepatoma cells by high-performance liquid chromatography

A rapid, specific, and sensitive method has been developed for the determination of ribonucleoside and deoxyribonucleoside triphosphates in Novikoff hepatoma cells. A simple three-step procedure was used. Extraction of the biological material with 5% cold trichloroacetic acid (TCA); elimination of TCA by ethilic ether wash and concentration of the sample by lyophilization; and separation of CTP, dCTP, ATP, dATP, UTP, dTTP, GTP, dGTP and their quantitation by anionic-exchange high-performance liquid chromatography under isocratic conditions. All the compounds were identified by comparing their retention times with those of pure compounds, by cochromatography with single pure ribonucleoside triphosphates (NTPs) or deoxyribonucleoside triphosphates (dNTPs), and by comparing the 280 nm:254 nm spectral ratios of the peaks with those of known NTP and dNTP standards. The specific activity of all the above mentioned nucleotides also was determined in Novikoff hepatoma cells labeled with [32P]orthophosphate.     

Sucrose metabolism in lima bean seeds

Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.     

Glycerol metabolism in higher plants: glycerol kinase

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or UTP could be used as the phosphoryl group donor. The Km for glycerol was 55 microM and Km values for the nucleoside triphosphates were 145-620 microM. The Vmax for the reaction was 40-78 pmol product per min. Kinetic data indicate that the enzyme has a sequential mechanism.     

Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Hormone and seed-specific regulation of pea fruit growth

Growth of young pea (Pisum sativum) fruit (pericarp) requires developing seeds or, in the absence of seeds, treatment with gibberellin (GA) or auxin (4-chloroindole-3-acetic acid). This study examined the role of seeds and hormones in the regulation of cell division and elongation in early pea fruit development. Profiling histone H2A and gamma-tonoplast intrinsic protein (TIP) gene expression during early fruit development identified the relative contributions of cell division and elongation to fruit growth, whereas histological studies identified specific zones of cell division and elongation in exocarp, mesocarp, and endocarp tissues. Molecular and histological studies showed that maximal cell division was from -2 to 2 d after anthesis (DAA) and elongation from 2 to 5 DAA in pea pericarp. Maximal increase in pericarp gamma-TIP message level preceded the maximal rate of fruit growth and, in general, gamma-TIP mRNA level was useful as a qualitative marker for expanding tissue, but not as a quantitative marker for cell expansion. Seed removal resulted in rapid decreases in pericarp growth and in gamma-TIP and histone H2A message levels. In general, GA and 4-chloroindole-3-acetic acid maintained these processes in deseeded pericarp similarly to pericarps with seeds, and both hormones were required to obtain mesocarp cell sizes equivalent to intact fruit. However, GA treatment to deseeded pericarps resulted in elevated levels of gamma-TIP mRNA (6 and 7 DAA) when pericarp growth and cell enlargement were minimal. Our data support the theory that cell division and elongation are developmentally regulated during early pea fruit growth and are maintained by the hormonal interaction of GA and auxin.     

Proteinase activities in resting and germinating seeds of Scots pine, Pinus sylvestris

Resting seeds of Scots pine contained a moderate amount of acid proteinase activity, about 90% of which was inhibited by pepstatin A and about 10% by p-hydroxymer-curibenzoate. In gel chromatography on Sephacryl S-200 the proteinase activity showed a complex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which are the main proteinases in the endosperms of germinating seeds (Salmia 1981: Physiol. Plant. 51: 253–258), were not present in the resting seeds.—Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200. As none of the peaks had the catalytic properties of proteinase I or II, it seems that these endospermal enzymes are also lacking in the seedling tissues.—In the endosperms of germinating seeds the activity of the pepstatin-sensitive acid proteinase(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage.—Resting embryos contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hy-droxymercuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases increased continuously with the growth of the seedling.—It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and II, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm.     

Chemical Examination of Viable and Non-Viable Rice Seeds

A chemical examination has revealed four phenolic compounds in nonviable rice seeds (Oryza sativa L.). Their inhibitory bioactivity in the wheat coleoptile extension bioassay and the absence of these components in viable seeds have confirmed the relation of the phenolics with nonviability of the seeds. The compounds isolated from nonviable rice seeds were Kaempferol-4′-methyl ether, Kaempferol, quercetin and caffeic acid as identified on the basis of physical and chemical evidences.     

The 26 Nudix hydrolases of Bacillus cereus, a close relative of Bacillus anthracis

The genome of Bacillus cereus contains 26 Nudix hydrolase genes, second only to its closest relative, Bacillus anthracis which has 30. All 26 genes have been cloned, 25 have been expressed, and 21 produced soluble proteins suitable for analysis. Substrates for 16 of the enzymes were identified; these included ADP-ribose, diadenosine polyphosphates, sugar nucleotides, and deoxynucleoside triphosphates. One of the enzymes was a CDP-choline pyrophosphatase, the first Nudix hydrolase active on this substrate. Furthermore, as a result of this and previous work we have identified a new sub-family of the Nudix hydrolase superfamily recognizable by a specific amino acid motif outside of the Nudix box.     

The Assimilation of Ureides in Shoot Tissues of Soybeans : 1. CHANGES IN ALLANTOINASE ACTIVITY AND UREIDE CONTENTS OF LEAVES AND FRUITS

The ureides, allantoin and allantoic acid, are major forms of N transported from nodules to shoots in soybeans (Merr.). Little is known about the occurrence, localization, or properties of the enzymes involved in the assimilation of ureides in shoot tissues. We have examined the capacity of the shoot tissues to assimilate allantoin via allantoinase (EC 3.5.2.5) during leaf and fruit development in nodulated soybeans. Specific activity of allantoinase in leaves peaked during pod formation and early seed filling. In developing fruits allantoinase activity in the seeds was 2 to 4 times that in the pods when expressed on a fresh weight or organ basis. In seeds, the embryos contained the highest specific allantoinase activity. Stems and petioles also had appreciable allantoinase activity. With development, peaks in the amounts of allantoic acid, but not allantoin, were measured in both leaves and fruits suggesting that the assimilation of allantoic acid may be a limiting factor in ureide assimilation. Highest amounts of ureides were measured in the pith and xylem of stem tissues and in developing pod walls.     

Studies on the proteins from the seeds of Croton tiglium and of Jatropha curcas. Toxic properties and inhibition of protein synthesis in vitro.

1. Proteins extracted from the seeds of the Euphorbiaceae croton tiglium and Jatropha curcas were separated into three major peaks (I,II,and III) by Sephadex chromatography. 2. The crude protein from both seeds and peaks I and II from Croton and peak I from Jatropha were toxic to mice, to different extents. 3. The crude protein and peak I and peak II from both seeds, inhibited protein synthesis by a reticulocyte lysate; maximum inhibition was exerted by peak II from both seeds. None of these preparations affected protein synthesis in vitro by Ehrlich ascites cells.     

Growth, sucrose synthase, and invertase activities of developing Phaseolus vulgaris L. fruits

Activities of the sucrose-cleaving enzymes, acid and neutral invertase and sucrose synthase, were measured in pods and seeds of developing snap bean (Phaseolus vulgaris L.) fruits, and compared with ¹⁴C-import, elongation and dry weight accumulation. During the first 10 d post-anthesis, pods elongated rapidly with pod dry weight increase lagging behind by several days. The temporal patterns of acid invertase activity and import coincided closely during the first part of pod development, consonant with a central role for this enzyme in converting imported sucrose during pod elongation and early dry weight accumulation. Later, sucrose synthase became the predominant enzyme of dry weight accumulation and was possibly associated with the development of phloem in pod walls. Sucrose synthase activity in seeds showed two peaks, corresponding to two phases of rapid import and dry weight accumulation; hence, sucrose synthase was associated with seed sink growth. Acid invertase activities in seeds were low and did not show a noticeable relationship with import or growth. All neutral invertase activities, during pod and seed development, were too low for it to have a dominant role in sucrose cleavage. Changes in activities of certain sucrose-cleaving enzymes appear to be correlated with certain sink functions, including import, storage of reserves, and biosynthetic activities. The data supports the association of specific sucrose-cleaving enzymes with the specific processes that occur in the developing pods and seeds of snap bean fruits; for example, acid invertase with pod elongation and sucrose synthase with fruit dry matter accumulation.     

Analysis of Indole-3-Acetic Acid Metabolites from Dalbergia dolichopetala by High Performance Liquid Chromatography-Mass Spectrometry

A mixture of [2-¹⁴C₁] and [¹³C₆]indole-3-acetic acid was applied to the cotyledons of 6-day-germinated seeds of “jacarandá do cerrado” (Dalbergia dolichopetala) and after 8 hours the seeds were extracted. Analysis of the fractionated extract by reversed-phase high performance liquid chromatography-radiocounting revealed the presence of five radiolabeled metabolite peaks (I-V). After further purification, the individual peaks of radioactivity were analyzed by combined high performance liquid chromatography-steel filter-fast atom bombardment-mass spectrometry. The metabolite fraction V was found to contain [¹⁴C₁, ¹³C₆]indole-3-acetylas-partic acid and unlabeled indole-3-acetylglutamic acid. Analysis of the metabolite fraction II revealed the presence of dioxindole-3-acetylaspartic acid and putative dioxindole-3-acetylglutamic acid as well as putative benzene ring-hydroxylated derivatives of oxindole-3-acetylaspartic acid and oxindole-3-acetylglutamic acid. There was no evidence of significant incorporation of label from [2′-¹⁴C₁] or [¹³C₆]indole-3-acetic acid into any of these conjugated indoles.     

Identification of gibberellins A17, A25, A45, abscisic acid,phaseic acid,and dihydrophaseic acid in seeds of Pyrus communis

Gibberellin A₁₇, abscisic acid, and 4′-dihydrophaseic acid were identified by GC-MS of derivatized extracts from both immature and mature seeds of pear. Immature seeds also contained phaseic acid, gibberellins A₂₅ and A₄₅, and two presumed mono-hydroxylated derivatives of GA₄₅, one of which was tentatively identified as 3β-hydroxy-GA₄₅. Several presumed metabolites of abscisic acid were detected in both mature and immature seeds.     

An allelopathic substance exuded from germinating watermelon seeds

When watermelon seeds were cultured in a Petri dish together with amaranth, barnyard grass, cockscomb, lettuce or tomato seeds, the shoot growth of amaranth and cockscomb was markedly promoted, whereas the shoot growth of lettuce and tomato was inhibited. The shoot growth of barnyard grass was not affected. These results suggest that plant-selective allelopathic substance(s) affecting the shoot growth of other plant seedlings were exuded from watermelon seeds. An allelopathic substance was isolated from the exudates of germinating watermelon seeds and identified as vanillic acid by its spectral analysis and Rf value on TLC. Vanillic acid promoted the shoot growth of cockscomb at the concentrations of 300 to 10 mg/l and that of amaranth at the concentrations of 30 to 3 mg/l, although the shoot growth of amaranth was inhibited by 300 mg/l of vanillic acid. The shoot growth of lettuce and tomato was inhibited at the concentrations higher than 30 mg/l by vanillic acid. However, the shoot growth of barnyard grass was not affected at the concentrations used. All these results suggest that vanillic acid may play as a major component of allelopathic substance(s), which shows plant-selective activity, in the exudates of germinating watermelon seeds.     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液(pH4.0),1%PAA选择性沉淀碱性蛋白(等电点pI为8.65~9.30)的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA(1%)用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH>9.0),当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经SephadexG-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.     

猫儿屎和三叶木通种子油中脂肪酸成分的GC-MS分析

采用索氏提取法提取木通科植物猫儿屎和三叶木通种子的脂溶性成分,甲酯化处理后用气相色谱-质谱联用技术(GC-MS)分离和鉴定其组成和含量。从猫儿屎种子油中鉴定出9种脂肪酸,占检出物总质量分数的94.67%(其中饱和脂肪酸占12.63%,不饱和脂肪酸占82.04%),主要成分为9-十六烯酸(47.22%)、9-油酸(27.13%)、棕榈酸(10.75%)、亚油酸(7.47%)和硬脂酸(1.61%)。从三叶木通种子油中鉴定出10种脂肪酸,占检出物总质量分数的99.75%(其中饱和脂肪酸占23.39%,不饱和脂肪酸占76.36%),主要成分为11-油酸(47.63%)、亚油酸(27.05%)、棕榈酸(20.14%)、16-甲基-十七烷酸(3.03%)和8-油酸(1.07%)。结果表明,猫儿屎和三叶木通种子油中脂肪酸含量丰富,在食用、医疗保健等方面具有较高的应用潜力和综合开发前景。