Chemical Modification of Tryptophan Residues in Castor Bean Hemagglutinin

Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that the extent of modification of tryptophan in CBH is influenced by an ionizable group with pK_a = 3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-chain of CBH.     

Identification of the Tryptophan Residue Located at the Saccharide Binding Site of Castor Bean Hemagglutinin

The tryptophan residue present at the saccharide-binding site of castor bean hemagglutinin (CBH) was identified. A peptide containing a modified tryptophan residue was isolated from the tryptic digest of S-car boxy methylated B-chain obtained from an inactive derivative of CBH (2-Oxa-CBH), in which two tryptophan residues/mol were oxidized with Af-bromosuccinimide, by gel filtration on a Sephadex G-50 followed by high performance liquid chromatography. Analytical data for the isolated peptide indicated that the tryptophan residue at position 131 on the B-chain was modified in 2-Oxa-CBH.

From these and earlier results, it is suggested that the tryptophan residue at 131 on each B-chain is closely associated with the saccharide-binding activity of CBH. The specific role of tryptophan residue at 131 in the saccharide-binding site of CBH is also discussed.     

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

Expression of Phospholipase D during Castor Bean Leaf Senescence

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.     

Sucrose-Induced Increase of Invertase Synthesis in Castor Bean Cotyledons

Abstract

Aumento della sintesi di invertasi in seguito a trattamento con saccarosio in cotiledoni isolati da semi germinanti di ricino. – L'attività invertasica per cotiledone aumenta durante la germinazione di semi di ricino. In cotiledoni isolati ed incubati in acqua distillata per 15–22 ore, l'aumento di attività invertasica è molto scarso, L'aggiunta di saccarosio 0,1 M al mezzo di incubazione provoca un aumento di circa 40% dell'attività invertasica; aumento che non si riscontra se i cotiledoni vengono incubati in glucosio 0,1 M. La pre-senza di attinomicina D e di puromicina nel mezzo di incubazione previene lo sviluppo dell'attività invertasica. L'apparente specificità del saccarosio nell'indurre l'aumento di sintesi dell'enzima viene brevemente discussa nel quadro piú ampio dei fenomeni di regolazione da substrato delle sintesi di enzimi.     

蓖麻提取物对鼠抗生育作用的实验研究

利用蓖麻提取物对昆明种小鼠进行了短期与长期的抗生育实验,研究发现蓖麻提取物（蓖麻油和蓖麻蛋白）对小鼠有明显的抗生育作用。蓖麻蚩白及其与蓖麻油的混合物在抗早孕方面的效果均可达到100％,蓖麻油抗着床的效果也可达到100％。蓖麻油长期抗鼠生育效果明显,在210d（正常小鼠的妊娠期是21～23d）内有效降低小鼠生育代数与产仔数,生育抑制率达80％以上。蓖麻提取物对离体小鼠子宫的影响也非常显著,通过增强小鼠子宫内部收缩有效减少着床机率。在中止妊娠的实验中发现,服用了蓖麻蛋白及其与蓖麻油的混合物的小鼠子宫内没有着床位点。     

Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.     

Arsenic Speciation in Phloem and Xylem Exudates of Castor Bean

How arsenic (As) is transported in phloem remains unknown. To help answer this question, we quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)], or dimethylarsinic acid. In the As(V)- and As(III)-exposed plants, As(V) was the main species in xylem exudate (55%–83%) whereas As(III) predominated in phloem exudate (70%–94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray mass spectrometry coupled to high-performance liquid chromatography identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no As(III)-thiol complexes. It is thought that As(III)-thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, dimethylarsinic acid was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results show that free inorganic As, mainly As(III), was transported in the phloem of castor bean exposed to either As(V) or As(III), and that methylated As species were more mobile than inorganic As in the phloem.Arsenic (As) is an environmental and food chain contaminant that has attracted much attention in recent years. Soil contamination with As may lead to phytotoxicity and reduced crop yield (Panaullah et al., 2009). Food crops are also an important source of inorganic As, a class-one carcinogen, in human dietary intake, and there is a need to decrease the exposure to this toxin (European Food Safety Authority, 2009). Paddy rice (Oryza sativa) is particularly efficient in As accumulation, which poses a potential risk to the population based on a rice diet (Meharg et al., 2009; Zhao et al., 2010a). Other terrestrial food crops generally do not accumulate as much As as paddy rice; however, where soils are contaminated, relatively high concentrations of As in wheat (Triticum aestivum) grain have been reported (Williams et al., 2007; Zhao et al., 2010b). On the other hand, some fern species in the Pteridaceae family are able to tolerate and hyperaccumulate As in the aboveground part to >1,000 mg kg⁻¹ dry weight (e.g. Ma et al., 2001; Zhao et al., 2002); these plants offer the possibility for remediation of As-contaminated soil or water (Salido et al., 2003; Huang et al., 2004). A better understanding of As uptake and long-distance transport, metabolism, and detoxification is needed for developing strategies for mitigating As contamination, through either decreased As accumulation in food crops or enhanced As accumulation for phytoremediation.The pathways of As uptake by plant roots differ between different As species; arsenate [As(V)] enters plant cells via phosphate transporters, whereas arsenite [As(III)] is taken up via some aquaporins (for review, see Zhao et al., 2009). In rice, a silicic acid efflux protein also mediates As(III) efflux toward stele for xylem loading (Ma et al., 2008). Methylated As species, such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)], which may be present in the environment as products of microbial or algal methylation of inorganic As or from past uses of methylated As pesticides, are taken up by rice roots partly through the aquaporin NIP2;1 (for nodulin 26-like intrinsic protein; also named Lsi1; Li et al., 2009). Once inside plant cells, As(V) is reduced to As(III), possibly catalyzed by As(V) reductase(s) such as the plant homologs of the yeast (Saccharomyces cerevisiae) ACR2 (Bleeker et al., 2006; Dhankher et al., 2006; Ellis et al., 2006; Duan et al., 2007). As(III) has a high affinity to thiol (-SH) groups and is detoxified by complexation with thiol-rich phytochelatins (PCs; Pickering et al., 2000; Schmöger et al., 2000; Raab et al., 2005; Bluemlein et al., 2009; Liu et al., 2010). As(III)-PC complexation in roots was found to result in reduced mobility for efflux and for long-distance transport, possibly because the complexes are stored in the vacuoles (Liu et al., 2010). Excess As(III) causes cellular toxicity by binding to the vicinal thiol groups of enzymes, such as the plastidial lipoamide dehydrogenase, which has been shown to be a sensitive target of As toxicity (Chen et al., 2010). The As hyperaccumulating Pteris species differ from nonhyperaccumulating plants because of enhanced As(V) uptake (Wang et al., 2002; Poynton et al., 2004), little As(III)-thiol complexation (Zhao et al., 2003; Raab et al., 2004), and efficient xylem loading of As(III) (Su et al., 2008). Recently, an As(III) efflux transporter, PvACR3, has been found to play an important role in As(III) detoxification by transporting As(III) into vacuoles in Pteris vittata (Indriolo et al., 2010).With the exception of As hyperaccumulators, most plant species have a limited root-to-shoot translocation of As (Zhao et al., 2009). The chemical species of As in xylem exudate have been determined in a number of plant species. As(III) was found to be the predominant species (80%–100%) in the xylem sap of rice, tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and P. vittata even when these plants were fed As(V) (Mihucz et al., 2005; Xu et al., 2007; Ma et al., 2008; Su et al., 2010), suggesting that As(V) is reduced in roots before being loaded into the xylem. In other plant species, such as Brassica juncea (Pickering et al., 2000), wheat, and barley (Hordeum vulgare; Su et al., 2010), As(V) accounted for larger proportions (40%–50%) of the total As in the xylem sap. Studies using HPLC-inductively coupled plasma (ICP)-mass spectrometry (MS) coupled with electrospray (ES)-MS showed no evidence of As(III)-thiol complexation in the xylem sap of sunflower (Helianthus annuus; Raab et al., 2005). When rice plants were exposed to MMA(V) or DMA(V), both As species were found in the xylem sap (Li et al., 2009). Generally, methylated As species are taken up by roots at slower rates than inorganic As, but they are more mobile during the xylem transport from roots to shoots (Marin et al., 1992; Raab et al., 2007; Li et al., 2009).It has been shown that phloem transport contributes substantially to As accumulation in rice grain (Carey et al., 2010). However, little is known about how As is transported in phloem (Zhao et al., 2009). There are no reports on the chemical species of As in phloem exudate. The speciation of As in phloem is important because it dictates how As is loaded in the source tissues and unloaded in the sink tissues, such as grain. Questions with regard to the oxidation state, methylation, and complexation of As in phloem sap remain to be answered. Unlike xylem sap, phloem sap is much more difficult to obtain in sufficient quantities for analysis. In this study, we investigated As speciation in phloem and xylem exudates of castor bean (Ricinus communis), which is widely used as a model plant to investigate phloem transport of solutes (e.g. Hall et al., 1971; Hall and Baker, 1972; Allen and Smith, 1986; Bromilow et al., 1987).     

N-Acetylglucosamine Transfer Reactions and Glycoprotein Biosynthesis in Castor Bean Endosperm

N-Acetyl-[³H]glucosamine supplied to intact 3 d old castor beanendosperm tissue was incorporated into TCA-insoluble productpresumed to be glycoprotein. After an incubation time of 2 hthe major paniculate location of this product within the cellwas the endoplasmic reticulum. Cell-free preparations containingparticulate enzymes transferred N-acetyl-[¹⁴C]glucosamine fromUDP-N-acetyl-[¹⁴C]glucosamine into a fraction soluble in chloroform/methanol(2: 1, by vol), a fraction soluble in chloroform/methanol/water(10: 10: 3, by vol.), and an insoluble residue. Mild acid hydrolysisreleased the saccharide moieties from the lipids. Paper chromatographicanalysis of the released saccharides established that the C/M-solubleproducts contained both N-acetyl-[¹⁴C]glucosamine and N, N'-diacetyl-[¹⁴C]chitobiose.In contrast, N-acetyl-[¹⁴C]glucosamine released from the C/M/W-solubleproduct was contained in an oligosaccharide, probably in associationwith unlabelled mannose residues. The stimulatory effect ofdolichol monophosphate and the inhibitory effect of tunicamycinon saccharide-lipid synthesis indicated that N-acetyl-glucosamineis transferred to a glycopolymer by the established reactionsof the dolichol monophosphate pathway. The enzymes catalysingthe constituent reactions of this pathway were exclusively locatedin the ER.     

Deterioration of Mitochondrial Membranes from Castor Bean Endosperm by Hydrogen Peroxide

Loss of membrane integrity by hydrogen peroxide (H₂O₂) was studiedin isolated mitochondria of castor bean. Incubation of mitochondriawith H₂O₂ resulted in the release of fumarase with a concomitantloss of phospho-lipids. Degradation of membrane was found tooccur independent of lipid peroxidation. H₂O₂-treated mitochondriawere able to degrade exogenous radiolabelled phospho-lipids. ³Corresponding author; fax 82-42-821-2391     

蓖麻杂交种的SSR鉴定及遗传变异分析

采用SSR标记对蓖麻CSR24×CSR181杂交所得的F1种子进行分析,为蓖麻早期杂种鉴定和遗传变异分析奠定技术与理论基础。结果表明:(1)各位点鉴定所得结果基本一致,除RCM207和RC129位点鉴定的杂种率未超过10%外,其它位点的杂种率都十分接近,在13.46%～17.27%之间。(2)少数个体在相关位点发生了变异,在引物RC242的扩增图谱中有4个单株出现了双亲特异条带的缺失,产生了双亲都没有的新条带;在引物Rco23、Rco26、Rco29、RC129、RCM613和RCM999的扩增结果中出现了父本特异条带的缺失,同时产生了一条新条带。(3)多样性及UPGMA聚类分析表明杂交后代遗传变异显著,子代个体与亲本间的遗传相似系数在0.45～1.0之间,个体间差异明显。     

Acid Lipase of Castor Bean Lipid Bodies : Isolation and Characterisation

The acid lipase of castor endosperm lipid bodies has been studied using colorimetric assay based on the measure of the hydrolytic activity of p-nitrophenyl ester of palmitate and other acyl derivatives. These substrates are compatible with the natural triacylglycerols for the measure of lipolytic activities. The subcellularly-surveyed acid lipolytic activity in the germinated castor bean endospermal tissue was found to be enhanced in the lipid bodies. The lipase, which is partially latent and tightly associated with lipid bodies, is an exceptionally stable enzyme with an optimum activity at pH 4.5 and displays an inverse relationship between its activity and the acyl chain length of its substrate. To facilitate isolation of the acid lipase, a procedure has been developed to solubilise the membrane-bound enzyme in an active form. The detergent-solubilised acid lipase after two chromatographic steps yielded an eight-fold active preparation which after gel permeation resolved as heterogeneous aggregate in excess of 500 kD. Lipase-enriched preparations showed consistent presence of 14 and 60 kD proteins which constituted the most abundant species of the lipid bodies. Although it has not been possible to obtain an active lipase preparation in a state free of either the 14 or 60 kD protein, the lipase activity in the detergent extracts of lipid bodies was immunoprecipitable with antibodies raised against the 60 kD component.     

蓖麻油微胶囊化及对鼠抗生育研究

以甲苯2,4-二异氰酸酯(TDI)和乙二醇为原料,采用界面聚合法对蓖麻油进行微胶囊化,制成蓖麻油微胶囊。通过正交试验、显微镜计数法、测微尺测量其微胶囊直径等方法来研究影响微胶囊大小、分布和包埋率的各种因素,寻求制备微胶囊的最佳工艺条件,并利用制得的蓖麻油微胶囊对昆明种小鼠进行抗生育实验。实验结果表明:优化后的蓖麻油包埋率达95%以上,其直径分布在4~120μm之间,平均20μm。蓖麻油微胶囊抗着床的有效率可达80%以上,其中200 mg/kg剂量组为100%。抗早孕的效果也可达73%以上,其中400 mg/kg组为100%。