Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.     

苹果果实糖积累特性与品质形成的关系

以'富士'和'国光'苹果为研究对象,对其果实发育过程中糖含量及其代谢关键酶活性的变化进行测定分析,以揭示糖分积累代谢特性对果实品质形成的影响.结果表明:(1)'富士'和'国光'均为己糖积累型果实, '富士'果实以积累果糖最多,果糖/葡萄糖(F/G)值为1.56,而'国光'以积累葡萄糖最多,F/G值仅为0.68;蔗糖在两品种中含量和所占比例均很低,在近成熟期'富士'高于'国光'.(2)'富士'果实蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)活性均随果实糖的累积量增加而显著升高,酸性转化酶(AI)活性也渐趋升高,而中性转化酶(NI)活性波动不大,且其糖累积与AI和SPS活性相关性最大,而与NI相关性不大,SS的作用主要表现在发育后期;在 '国光'果实糖积累过程中SPS起主导作用,SS和NI的作用主要表现在发育前期,而AI的作用不大.(3)'富士'和'国光'果实淀粉含量变化趋势相同,在淀粉积累高峰之后,'富士'果实淀粉降解速度更快,其淀粉含量迅速下降且低于'国光',此时其相应淀粉酶活性也高于'国光'.研究发现,'富士'和'国光'果实糖积累和淀粉代谢均存在显著差异,从而直接或间接地影响着果实糖代谢过程,进而导致果实品质的显著差异.     

Kiwellin, a Novel Protein from Kiwi Fruit. Purification, Biochemical Characterization and Identification as an Allergen*

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit. *The protein sequence data reported in this paper will appear in the Swiss-Prot and TrEMBL knowledgebase under the accessionnumber P84527.     

苹果果实香气产生过程中氨基酸和脂肪酸含量及一些相关酶活性的变化

研究了苹果果实成熟期间香气和乙烯的产生动态,以及游离氨基酸、游离脂肪酸含量和脂氧合酶(LOX)、醇-酰基转移酶(AAT)活性的变化.结果表明,果实香气物质是随着乙烯释放的增加而产生和增加的.在此过程中,异亮氨酸大量积累.游离脂肪酸在果实香气很少时呈增加趋势;随着香气产生的增多而迅速下降;乙烯高峰过后又有增加.脂氧合酶活性随着果实成熟而提高,其活性在乙烯释放达到高峰时达到最大值,之后迅速下降.醇-酰基转移酶活性在果实开始产生香气时迅速增加,之后保持较高活性.     

Sunburn of Apple Fruit: Historical Background,Recent Advances and Future Perspectives

Sunburn is a physiological disorder of apples and other fruit species caused by excess solar radiation. Damage occurs in practically all growing regions of the world, causing severe crop loss every year. Direct factors required for induction of the three currently-known types of sunburn (i.e., sunburn necrosis, sunburn browning, and photooxidative sunburn) include excess radiant heating and/or exposure to excess sunlight. Several other factors (e.g., relative humidity, wind velocity, acclimation of fruit, and cultural management practices), which alone cannot induce sunburn damage, indirectly influence the induction of sunburn by interacting with the direct factors to influence the appearance and severity of the symptoms. Sunburn affects apple fruit at many levels; it causes structural and morphological changes, alters pigment composition, influences adaptive mechanisms, impairs photosynthesis, and consequently decreases fruit quality. Fruits employ multiple physiological and biochemical mechanisms as complex defense systems to minimize damage. Photoprotective pigments, antioxidant enzymes and metabolites, heat-shock proteins, and the xanthophyll cycle help mitigate damage, but are often inadequate under field conditions to fully protect from sunburn. Quality loss significantly affects postharvest behavior, marketing and consumer acceptance of fruit. Internal fruit quality (e.g., firmness, soluble solids concentration, and titratable acidity) is affected by sunburn, and changes in these traits continue during cold storage. Sunburn-related disorders (e.g., sunburn scald in ‘Granny Smith’ and ‘Fuji’ stain) can appear in cold storage. There are several methods with various modes of action (e.g., climate ameliorating techniques, and sunburn suppressants) available to growers to decrease sunburn under field conditions. At the end of this review, the potential impact of a changing climate on sunburn incidence is considered, as both UV-B radiation and temperature are projected to change. Finally, several topics that need further research are discussed.     

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Soluble invertase was purified from pea(Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation,DEAE-Sepharose column,Con-A-and Green 19-Sepharose affinity columns,hydroxyapatite column,ultra-filtration,and Sephacryl 300 gel filtration.The purified soluble acid(SAC) and alkaline(SALK) invertases had a pH optimum of 5.3 and 7.3,respectively.The temperature optimum of two invertases was 37 ℃.The effects of various concentrations of Tris-HCl,HgCl2,and CuSO4 on the activities of the two purified enzymes were examined.Tris-HCl and HgCl2 did not affect SAC activity,whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK activity by about 50%.SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 by 50%,respectively.The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis.The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM,respectively.The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa.The molecular mass of SALK was 30 kDa.Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7,respectively.     

番茄果实中焦磷酸:D-果糖-6-磷酸 1-磷酸转移酶的两种酶型的分离纯化及其特性

用快速蛋白液相层析仪(FPLC)Mono Q柱(HR5/5)分离纯化成熟绿番茄果实中PFP的两种分子酶型及其特性。一种酶型为Q_1,是含两个β-亚基(60kD)的二聚体,比活为5μmol min~(-1) mg~(-1);另一种为Q_2,由四个α-亚基(66kD)和四个β-亚基(60kD)组成八聚体,比活为70.5μmol/min~(-1)·mg~(-1)。Q_1的分子量是120kD,Q_2的分子量介于500kD和530kD之间。用纯化的Q_2制备的抗血清专一地与Q_2起沉淀反应。PFP酶液贮存后,其Q_1/Q_2蛋白量比值增加明显,表明部分Q_2转化为Q_1。Q_1具有催化活力表明PFP的活性中心位于β-亚基。α-亚基可能借增强PFP酶对F2,6P_2的亲和力以提高酶的比活而起调节功能,但是Q_1的活力依赖于F2,6P_2的激活,表明β-亚基处也可能存在F_2,_6P_2的调节位点。Q_2含紧密结合的F2,6P_2分子,并表现出对F2,6P2_的不敏感性,基于此种现象,有必要重新认识PFP对F2,6P_2敏感性的内在实质。     

高分子量麦谷蛋白１４和１５亚基的纯化、Ｎ—末端序列及部分生化特性研究

用ＳＤＳ－ＰＡＧＥ制备电泳技术结合一种新的凝胶中蛋白质显色方法,对普通小麦（Triticum aestivum)小偃六号的高分子量麦谷蛋白１４和１５亚基进行了有效的分离纯化,将其转印于ＰＶＤＦ膜上测定了Ｎ－端的氨基酸顺序,通过比较了发现它们与已知序列的其他的高分子是麦谷蛋白亚基高度同源。用两种双向电泳技术确定了它们的等电点（ＰＩ）属于碱性范围。     

豌豆铁蛋白的纯化及其抗血清的制备

干豌豆种子粗提物经MgCl2 盐析、AcA2 2 凝胶过滤和DEAE 纤维素阴离子交换柱层析等方法进行纯化 ,以邻菲咯啉显色法检测铁蛋白 ,最后获得纯的铁蛋白 .纯化的铁蛋白在PAGE上显示一条带 ,SDS PAGE显示该蛋白仅含 2 8kD一条亚基 .纯化的豌豆铁蛋白免疫兔 7周后 ,琼脂糖双扩散法检测抗血清效价达 1∶32 .用分级盐析法纯化抗血清 ,纯化后的抗体用琼脂糖双扩散法对大豆铁蛋白粗提物有免疫交叉反应     

苦楝果多糖的分离纯化及组成分析

利用水提醇沉法提取苦楝果实中的多糖,经DEAE-52柱层析分离,得到MP1、MP2和MP3三个多糖组分,用Sephadex G-100凝胶色谱柱对MP1进行纯化鉴定,结果显示为单一峰。借助气质联用仪,对苦楝粗多糖和组分MP1进行了成分分析。红外光谱分析表明苦楝多糖的单糖残基以吡喃环和呋喃环的形式存在。     

甘薯叶片蔗糖酶的分离纯化及其部分性质

纯化酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,ＳＤＳ－ＰＡＧＥ显示一条蛋自带,其亚基分子量为３９．８ｋＤ。用ＳｅｐｈａｃｒｙｌＳ－２００凝胶过滤测得全酶的分子量为７９．４ｋＤ,该酶由两个相同亚基组成。其表观Ｋｍ为１２ｍｍｏｌ／Ｌ,Ｖｍａｘ为９９．５ｍｇ还原糖ｍｇ－１ｐｒｏｔｅｉｎｈ－１。     

Relationship Between Growth, Secondary Metabolism, and Resistance of Apple

Abstract: The paper shows that N-induced vigorous shoot growth increases susceptibility of apple trees to Venturia inaequalis. This is due to a weakened defence in infected leaves of the high N cultures showing large lesions with excessive sporulation, whereas infected leaves from the low N cultures exhibited successful defence with only small chlorotic lesions and no sporulation. This might be explained by biosynthesis of phenylpropanoids in the young leaves of the resistant trees. A negative correlation between shoot growth of apple trees and the concentration of phenolic compounds in young leaves was found. Studies on in vitro shoot cultures revealed that the availability of sugars for the phenylpropanoid pathway is a strong regulatory factor. The ratio of sucrose and nitrogen in the medium influenced the total level of secondary products in the in vitro grown plantlets. Moreover, the relative deficiency of sugars was responsible for a metabolic block mainly at the level of glucosyl transferase and concomitant aglycone accumulation.     

Dormancy Release,Germination, and Electrolyte Leakage from Apple Embryos during Stratification in the Presence of Sucrose

The dynamics of dormancy release during the stratification of apple (Malus domestica Borkh.) seeds was quantitatively described by three characteristics of seeds germination: the percentage of seeds that germinated by the tenth day, mean germination time, and the sum of seeds germinated in each of ten days (Timson's parameter), which allowed the assessment of the viability, the rate of dormancy release, and seed heterogeneity. We showed that apple seeds were characterized by a combined (physical and physiological) type of dormancy, with the seed coat and the embryo envelope being involved in the maintenance of physical dormancy. The addition of sucrose to the stratification medium accelerated the release of seed dormancy and improved all characteristics that determine seed germinability. Electrolyte leakage from embryos hardly changed during stratification, which agrees with the fact that all seeds remained viable throughout the entire period of dormancy. We assume that the release of seed dormancy is not a single-stage process.     

Characterization of Protein Kinases in Apple and Grape During Fruit Development

The protein kinases were identified at different developmental stages of apple (Malus domestica L. Borth. cv. Red Starking) and grape (Vitis vinifera L. × V.lubrusca L. cv. Kyoho) fruit, and these protein kinases were found to be greatly activated by Ca2+. This demonstrated that calcium-dependent protein kinase plays an important role in apple and grape fruits. The results also showed that calmodulin (CaM) had no stimulation effect, but the CaM antagonists, such as calmidazolium, W7 and trifluoroacetic acid (TFA), had some inhibitory effects on the activities of this kind of protein kinase in both fruits, indicating that the detected protein kinases may be a kind of calmodulin-like domain protein kinase or calcium-dependent protein kinase (CDPK). There were distinct differences between apple and grape fruits, both in the characteristics and in the activity evolution patterns of the protein kinases during fruit development. The activity of calcium-dependent protein kinase was little changed in the three stages of apple fruit development, while in grape fruit the activity of this protein kinase was much higher in stage Ⅱ than those in stage Ⅰ and stage Ⅲ. The calcium-dependent protein kinase in apple fruits was strongly activated by Mn2+, and it was sensitive to heat treatment, but in grape fruits it was neither stimulated by Mn2+ nor sensitive to high temperature. Besides, phosphatidyl-serine (PS) had little effect on the activities of calcium-dependent protein kinase in grape fruits, but had stimulative effects in apple fruits. It was thus proposed that there might be a kind of calcium- and phosphatide-activated protein kinase, i.e., PKC, in apple fruits. The differences among the kinds, characteristics and the activity evolution patterns of protein kinases during apple and grape fruit development, suggest that the protein kinases may be involved in the process of fruit development.     

苹果和葡萄果实发育过程中蛋白激酶特性的比较研究

在苹果 (MalusdomesticaL .Borkh .cv .RedStarking)和葡萄 (VitisviniferaL .×V .lubruscaL .cv .Kyoho)的不同发育阶段均检测到蛋白激酶活性 ,它们可被Ca2 强烈激活 ,表明依赖钙的蛋白激酶是苹果和葡萄果实中一类非常重要的蛋白激酶。钙调素 (CaM)对苹果和葡萄果实蛋白激酶没有激活作用 ,同时CaM拮抗剂calmidazolium、W7及三氟乙酸 (TFA)均可不同程度地抑制其活性 ,表明依赖钙的蛋白激酶中可能有含类似钙调素结构域的蛋白激酶(CDPK)的存在。在苹果和葡萄果实发育中 ,依赖钙的蛋白激酶无论在其活性变化还是特性上都存在着显著差异 :苹果果实发育的不同阶段依赖钙的蛋白激酶活性无明显变化 ,而葡萄果实在发育的第Ⅱ期蛋白激酶活性大大高于第Ⅰ、Ⅲ期 ;苹果果实依赖钙的蛋白激酶可被Mn2 强烈激活并对热敏感 ,而葡萄果实依赖钙的蛋白激酶却不受Mn2 及热处理的影响 ;此外 ,葡萄果实蛋白激酶活性不受磷脂酰丝氨酸 (PS)的影响 ,而苹果果实蛋白激酶可被PS激活 ,表明苹果果实中还可能有钙和磷脂激活的蛋白激酶即PKC的存在。苹果和葡萄果实发育中蛋白激酶种类、特性及其活性变化的不同 ,暗示了这两种果实发育调控的差异 ,也意味着苹果和葡萄果实发育调控可能与蛋白激酶密切相关。     

SUA41蛋白表达和纯化及其多克隆抗体制备

旨在制备特异性SUA41多克隆抗体,为深入研究其在植物生长发育中的功能提供有力的分子生物学和生物化学的工具。PCR扩增拟南芥SUA41基因中编码280个氨基酸(401-680位氨基酸)的特异片段,经过GATEWAY的DNA重组技术构建了原核表达载体pDEST17-SUA41,用热休克法转化到E.coliBL21(DE3)star感受态细胞,以异丙基β-D-硫代半乳糖苷(IPTG)诱导表达出6×His-SUA41融合蛋白,用8 mol/L尿素缓冲液溶解包涵体并且经过水逐级去除尿素获得提纯的融合蛋白,并利用Western blotting鉴定确认。融合蛋白经Ni金属螯合柱亲和层析得以纯化,用SDS-PAGE进一步纯化。纯化的融合蛋白经过SDS-PAGE后切胶回收,免疫小白兔,制备多抗血清,然后用Western blotting进行检测,鉴定血清特异性和效价。结果显示,融合蛋白6×His-SUA41免疫兔,产生特异性的SUA41兔抗血清,可以检测到细菌和拟南芥组织中SUA41蛋白。用水提纯变性剂尿素溶解的包涵体蛋白具有可行性。制备的特异性SUA41兔抗血清效价高,能够有效地识别大肠杆菌表达的和拟南芥的SUA41蛋白。在有合适的对照情况下,该兔抗血清可以用于分析植物中SUA41蛋白的功能。     

枸杞果实铁型超氧物歧化酶的纯化及性质

枸杞果实Ｆｅ－ＳＯＤ粗提液经硫酸铵盐析、离子交换柱层析及凝胶过滤,纯化到电泳单班点均一程度。纯化的Ｆｅ－ＳＯＤ分子量为４４．６ｋＤ,亚基分子量为２２．０ｋＤ。金属元素分析表明,每分子酸含１个Ｆｅ原子。该酶在紫外区最大吸收值为２７８ｕｍ。Ｈ２Ｏ２明显抑制该酶活性,ＫＣＮ对酶活性无影响。该酶氨基酸组成与高等植物和蓝绿藻的Ｆｅ－ＳＯＤ相似,但它具有较高甘氨酸,酸性氨基酸与碱性氨基酸比值高于高等植物而与低等植物及原核生物相近。