昆虫神经毒性酯酶活性的测定

首次报道了针对昆虫建立的神经毒性酯酶(NTE)活性检测方法以及据此法所测得的棉铃虫幼虫的NTE活性。将测定脊椎动物NTE活性的方法改进并微量化以适用于无脊椎动物昆虫体内NTE活性测定。对于棉铃虫幼虫,该法测得其头部、中肠和脂肪体等3个部位的NTE活性分别为5.30,1.40和14.50nmolminmgprotein。     

几种杀虫剂对棉铃虫头部酯酶的联合抑制作用

测定了几种药剂及其组合对棉铃虫Helicoverpa armigera头部酯酶活性的独立与联合抑制作用。结果显示：久效磷、敌百虫、呋喃丹对酯酶活性抑制中浓度分别为:2.4443×10^-6、3.4562×10^-7、2.6302×10^-5mol/L。药代动力学分析结果显示:久效磷与呋喃丹对酯酶抑制方式为竞争性抑制,敌百虫为非竞争性抑制。久效磷+敌百虫与敌百虫+呋喃丹两种药剂组合处理后,对酯酶抑制均增强,抑制中浓度分别为:1.0846×10^-6、5.1786×10^-6mol/L,对酯酶的抑制作用属非竞争性抑制作用。而久效磷+呋喃丹组合对酯酶抑制活性相加,为竞争性抑制作用。结果说明杀虫剂与酯酶的药代动力学互作对混剂的酯酶的抑制作用方式和活性具有重要影响。     

黑翅土白蚁乙酰胆碱酯酶最佳反应体系的建立及药剂敏感度比较

用正交试验方法研究了酶浓度、底物浓度、反应体系pH值、反应温度、反应时间5个因素对黑翅土白蚁Odontotermes formosanus (Shiraki)乙酰胆碱酯酶(AChE)活性测定的影响。通过对正交试验结果进行极差和方差分析,明确了测定黑翅土白蚁AChE活性的最适反应条件是酶浓度为12.5 g/L,底物浓度为8 mmol/L,pH值8.0,反应温度40℃,反应时间5 min。此外,研究了6种药剂对黑翅土白蚁体内AChE活性的影响。结果表明：灭多威、辛硫磷、三唑磷、丙溴磷、马拉硫磷和氧化乐果6种药剂对黑翅土白蚁AChE抑制中浓度(IC₅₀)分别为3.52×10^-4,1.86×10^-3,5.13×10^-3,9.55×10^-4,8.81×10^-3,和1.39×10^-2 mol/L。在3.3×10^-7～5×10^-3 mol/L的浓度范围内,上述6种药剂对黑翅土白蚁体内AChE活性的抑制作用都具有明显的剂量效应关系。     

乙酰胆碱酯酶性质改变与昆虫抗药性的关系

乙酰胆碱酯酶是生物神经传导中的一种关键性酶,同时又是有机磷和氨基甲酸酯杀虫剂的靶标,因此一直是人们研究的热点。就近年来昆虫乙酰胆碱酯酶(AChE)在生化和分子生物学方面的研究进展、昆虫AChE基因结构及表达的变化对动力学参数、昆虫抗药性的影响机制以及害虫与天敌AChE的比较研究进行了简要综述。     

酶标板法监测棉蚜乙酰胆碱酯酶对杀虫剂的不敏感性

用酶标板法测定了不同浓度有机磷和氨基甲酸酯杀虫剂在反应不同时间内对棉蚜Aphis gossypii乙酰胆碱酯酶(AChE)的抑制作用。结果表明有机磷杀虫剂甲基1605,辛硫磷,久效磷,氧乐果和乙酰甲胺磷对棉蚜AChE均无明显的抑制作用。当用0.01 mol/L灭多威与酶及底物反应1 h对北京棉蚜(敏感)种群AChE的抑制率可达82.4%,与反应2 h 89.4%仅差7%。因此以0.01 mol/L灭多威反应1 h测定棉蚜AChE对它的敏感性是合理的。通过测定北京地区寄主为鼠李和棉花以及山东高密寄主为棉花的棉蚜种群中个体AChE活性的分布和灭多威对其抑制的分布,表明3个棉蚜种群中AChE个体频率的分布差异不大,而灭多威对三个种群个体AChE的抑制率小于30%的个体分别为2.4%、16%和29%。抑制率大于70%的个体分别为72%、33%和1%,与生物测定结果一致。因此,用酶标板法测定棉蚜个体AChE对氨基甲酸酯的不敏感性频率可作为棉蚜对氨基甲酸酯抗性的监测技术,为棉蚜化学防治提供依据。     

二化螟体内乙酰胆碱酯酶的分布及纯化方法

研究了二化螟Chilo suppressalis乙酰胆碱酯酶（AChE）的体躯和亚细胞分布,并用凝胶过滤层析和2种亲和层析方法从二化螟幼虫体内分离、纯化乙酰胆碱酯酶。结果表明：二化螟幼虫乙酰胆碱酯酶的活性主要集中于头部和胸部,而成虫胸部乙酰胆碱酯酶的活性最低,显著低于头部和腹部。成虫体内AChE的活性明显高于幼虫。在亚细胞的分布上,乙酰胆碱酯酶主要位于膜上（86%）,近46%的活性存在微粒体中。在3种纯化乙酰胆碱酯酶的方法中,以3-羧基苯基-乙基二甲基铵作配体的亲和层析法纯化效果最佳,乙酰胆碱酯酶的最高纯化倍数为536.05倍,产率30.46%。     

小菜蛾及菜蛾绒茧蜂乙酰胆碱酯酶敏感性的相关变化

用生物测定和生化检测的方法,对福州地区小菜蛾Plutella xylostella和菜蛾绒茧蜂Apanteles plutellae的抗药性及两种昆虫乙酰胆碱酯酶对杀虫剂的敏感性进行了田间监测。结果显示,从1998年9月至1999年4月,小菜蛾乙酰胆碱酯酶对6种有机磷和氨基甲酸酯杀虫剂敏感性逐渐恢复,寄生于同一虫源的菜蛾绒茧蜂乙酰胆碱酯酶敏感性的变化也呈明显的相关性,但菜蛾绒茧蜂乙酰胆碱酯酶的敏感性高于其寄主小菜蛾。脱离选择压力后,两种昆虫对杀虫剂的敏感性迅速恢复,乙酰胆碱酯酶的K_i值显著增高。对乙酰胆碱酯酶的K_m、V_max和K_i值测定结果表明,两种昆虫对有机磷和氨基甲酸酯杀虫剂的抗性与乙酰胆碱酯酶对杀虫剂的不敏感性有关。此外还研究了不同发育期小菜蛾乙酰胆碱酯酶活性及其K_i值的变化。探讨了在杀虫剂选择压力下,两种昆虫乙酰胆碱酯酶敏感性的环境适应性变化机制。     

六种常用杀虫剂对八种蚜虫的选择毒性

作者自1982年开始研究了乐果、氧化乐果、抗蚜威、氰戊菊酯、溴氰菊酯和氯氰菊酯等6种杀虫剂对8种蚜虫的选择毒性.以桃粉大尾蚜Hyalopterus amygdali Blanchard为标准,氧化乐果对桃粉大尾蚜和瓜蚜Aphis gossypii Glover之间的选择毒性指数最高为163.77,乐果和抗蚜威分别是373.24和34.70,而氰戊菊酯仅为1.37.氰戊菊酯最高的选择毒性指数是在桃粉大尾蚜和麦长管蚜Sitobionavenae(F.)之间,也只有6.86,有机磷和氨基甲酸酯杀虫剂对不同蚜虫的选择毒性与乙酰胆碱酯酶(AChE)对巯基试剂(DTNB)的敏感度有明显的相关性,说明其选择毒性与AChE的巯基结合部位有关.同时还发现,抗蚜威对洋槐蚜Aphis robiniae Macchiati和瓜蚜AChE的1₅₀值与其LC₅₀值表现一致.这些都说明了这两类杀虫剂对不同种蚜虫的选择毒性与AChE有关.氰戊菊酯和溴氰菊酯对蚜虫的选择毒性与α-乙酸萘酯羧酸酯酶的活性具有明显的相关性,而与β-乙酸萘酯羧酸酯酶的活性则无任何关系.氯氰菊酯的选择毒性与上述两种酯酶的活性没有任何相关性.     

淡色库蚊中抗性相关羧酸酯酶的纯化及其生化性质

在库蚊Culex pipiens品系中,非专一性酯酶活性的升高是对有机磷杀虫剂产生抗性的重要机理之一。应用SDS/PAGE,比较淡色库蚊Culex pipiens pallens抗敌百虫品系(RD)、敏感型品系(S)和抗苄呋菊酯品系(PY)中可溶性总蛋白质带型,显示RD中含有一条特异蛋白带,其它两个品系中未检出。在RD成虫匀浆液总蛋白中含量高达2.1%。分子量测定为66 kD。应用柱层析法分离得到了较纯的纯品。以α-NA为底物测得K_m=64.1 mmol/L,V_max=249.8 mmol/(L·mg·min)。与羧酸酯酶相比较：其K_m值小于已报道的抗性品系及非抗性品系A-酯酶和B-酯酶。V_max值比已报道抗性品系A-酯酶低,比B-酯酶高。较高浓度的敌百虫并不能抑制其酶活,属于A-酯酶。在昆虫体内可能主要通过结合隔离作用(sequestration)提高昆虫对有机磷的耐受性,对有机磷杀虫剂水解作用的可能性也不能排除。     

一种菊酯类复合剂对黑胸散白蚁体内CarEs和Ca-ATPase活性的影响

以黑胸散白蚁Reticulitermes chinensis Snyder为试验材料,研究了一种白蚁防治复合剂中的主要成分对白蚁体内羧酸酯酶（CarEs）和钙 腺苷三磷酸酶（Ca-ATPase）的影响。结果表明：氯菊酯在终浓度为1.66×10^-4 mol·L^-1以下时,对羧酸酯酶无明显抑制作用;八氯二丙醚和壬基酚聚氧乙烯醚在此浓度下都对羧酸酯酶表现出明显抑制作用,其IC₅₀分别为7.1148×10^-5 mol·L^-1和7.3373×10^-4 mol·L^-1;氯菊酯对Ca-ATPase表现出较强抑制作用,IC₅₀为5.11×10^-7 mol·L^-1。认为Ca-ATPase是黑胸散白蚁体内拟除虫菊酯类杀虫剂作用的主要靶标之一。     

金龟子绿僵菌MaYTTR-04菌株对松墨天牛成虫的致病力

松墨天牛Monochamus alternatus Hope是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。采用跗节接种法测定金龟子绿僵菌Metarhizium anisopliae MaYTTR-04菌株对松墨天牛成虫的致病力。结果表明： 在恒温条件下,松墨天牛第6天开始死亡,第18天～21天为死亡高峰期,每头成虫接种孢子量为2.3×10⁶±0.2×10⁶时成虫的死亡率第18天达85%,第21天达95%; 时间效应指标值LT₅₀为14.7天。室内自然变温条件下,松墨天牛第3天出现死亡,第15天～21天为死亡高峰期,每头成虫接种孢子量为2.3×10.6±0.2×10⁶时的死亡率在第15天达85%,第21天达100%;时间效应指标值LT₅₀为12.9天。林间套笼21天后,菌液处理的成虫平均死亡率为60%,僵虫率为48.9%;而通过无纺布菌条的成虫死亡率为86.7%,僵虫率为75.6%。表明MaYTTR-04菌株对松墨天牛成虫有强的致病力,且在较高变温环境温度下致病力更强。结果说明该菌株可作为生产性菌株应用于林间防治松墨天牛。     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

抑前胸腺肽在家蚕体内的活性作用

家蚕Bombyx mori抑前胸腺肽是昆虫脑神经肽的一种,体外实验表明它能抑制处于活动时期的家蚕前胸腺合成蜕皮激素,因此抑前胸腺肽可能对昆虫的变态起着重要的作用。将抑前胸腺肽以不同的浓度分单一注射和加强注射导入家蚕体内,不同的时间间隔取样,利用蜕皮激素放射免疫分析方法,观察到了抑前胸腺肽在家蚕体内的活性作用以及引起家蚕体内血淋巴中蜕皮激素浓度的动态变化,首次证明了抑前胸腺肽在体内对家蚕前胸腺合成蜕皮激素有强烈的抑制作用。     

Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots

Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10^–4 M and 1.1 x 10^–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg⁺. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )     

PENETRATION OF SUGARS FROM THE CHEMOSENSORY HAIR TIP IN THE FLESHFLY

(1) The penetrations of ¹⁴C-D-glucose and 3-O-methyl-¹⁴C-D-glucoseinto the isolated head of the fleshfly, Boettcherisca peregrina,from the tip of the longest chemosensory hair on the labrumwere investigated. (2) Both ¹⁴C-D-glucose and 3-O-methyl-¹⁴C-D-glucosepenetrated into the hair almost linearly with time and movedrapidly into the labrum. The isotopic activity was finally detectedin the head. (3) The isotopic activity of the hair dipped into¹⁴C-D-glucose solution increased in the preparation which hadbeen pretreated with 7.5 x 10^–2M colchicine for 30 min,whereas in the case of 3-O-methyl-¹⁴C-D-glucose no effect by7.5 x 10^–2M colchicine was found. (4) Both ¹⁴C-D-glucoseand 3-O-methyl-l4C-D-glucose which penetrated from the poreof the hair tip were detected in the dendritefree lumen andin the dendrite-containing lumen of the chemosensory hair. (5)These results suggest that D-glucose not only moves in the dendrite-freelumen and the dendrite-containing lumen but also in the dendrite(s).The suggestive results that 3-O-methyl-D-glucose moves in thedendrite(s) could not be obtained. ^*Present address: Department of Physiology, Medical Collegeof Miyazaki, Miyazaki, Japan.     

Some observations on the urea-degrading enzyme of the diatom Cyclotella cryptica and the role of nickel in its production

The urea-degrading enzyme of Cyclotella cryptica was testedin crude cell-free extracts for effects from chemical reagentsknown to distinguish between urease and ATP:urea amidolyase.Inhibition of the enzyme by hydroxyurea and its indifferenceto added ATP, Mg²⁺ or K⁺ avidin or biotin clearly characterizedthe enzyme as urease (EC 3.5.1.5 [EC] ). The Cyclotella urease wasunaffected by thiourea addition, as was also the growth of thediatom in the presence of this substrate analogue. Indirectevidence was obtained from growth studies of the diatom andcorresponding urease production showing that the enzyme: (i)contains Ni²⁺ tightly bound to an apoprotein; (ii) is producedconstitutively even from growth on nitrate and does not requireextracellular urea for its synthesis, although quantitativelythe activity is greatest from growth on urea. It is concludedthat Cyclotella urease is a Ni²⁺ constitutive enzyme similarin many respects to those previously reported from Phaeodactylumtricornutwn and Tetraselmis maculata.     

中华蜜蜂化学感受蛋白基因Acer-CSP1克隆与表达特征分析

化学感受蛋白(chemosensory proteins, CSPs)是昆虫化学感受系统中重要的组成部分之一。本研究克隆了中华蜜蜂Apis cerana cerana化学感受蛋白基因Acer-CSP1, 其核苷酸全长351 bp （GenBank登录号为FJ157352）, 编码116个氨基酸残基, 预测蛋白分子量为13.85 kD, 等电点为4.89, 且含有4个保守的半胱氨酸残基, 均符合昆虫CSPs的一般特征, 且与意蜂CSP1基因具有99.1%的相似性, 与其他昆虫也有45.3%~68.0%的相似性。利用2^-ΔΔCt法及绝对定量法的real-time PCR技术对Acer-CSP1在中蜂不同器官表达特征进行了研究, 得出的一致结论为Acer-CSP1显著水平地高丰度表达于中华蜜蜂触角, 其次大量表达于头部。由于触角为中华蜜蜂最主要的嗅觉器官, 而头部则具有发达的感觉神经系统和味觉系统, 这也提示Acer-CSP1极有可能参与中华蜜蜂的嗅觉以及其他化学感受功能。     

Purification and some properties of L-tyrosine carboxy-lyase from barley roots

L-Tyrosine carboxy-lyase (E. C. 4. 1. 1. 25) was extracted fromthe roots of barley seedlings and purified approximately 25fold. Optimum pH for the enzyme activity was found to be 7.3.The Km value for L-tyrosine was calulated as 4.5?10^–4M.D-Isomer did not react with the enzyme. L-DOPA, m-tyrosine ando-tyrosine were decarboxylated to some extent. Pyridoxal phosphateactivated the enzyme 4 fold. Caffeic acid and p-coumaric acidare competitive inhibitors. Ki values were 4.5?10^–5M forcaffeic acid and 1.6?10^–4M for p-coumaric acid. L-DOPAand m-tyrosine had an inhibitory effect on the decarboxylationof L-tyrosine. Hydroxylamine, semicarbazide, p-CMB, Fe⁺⁺, Cu⁺⁺,and Hg⁺⁺ inhibited the decarboxylation of tyrosine. Enzyme activitywas also found in extracts from Triticum aestivum, Zea maysand Cytisus scoparius. (Received November 30, 1973; )     

Differences in cold lability of pyruvate, Pi dikinase among C4 species

The cold lability of pyruvate, Pi dikinase in crude leaf extractswas studied in a number of C⁴ plants. The survey included C⁴monocots and dicots and species representingthe three C⁴ subgroups:NADP-malic enzyme, NAD-malic enzyme, and PEP-carboxykinase types. In some species (e.g., Digitaria sanguinalis, Sorghum bicolorand Echinochloa crus-galli), the enzyme was very sensitive tocold treatment (half life of about 8 min at 0?C and 10 to 15min at 10?C). In other species (Panicum miliaceum, Panicum maximumand Panicum texanum), the enzyme was very cold tolerant (retentionof 60 to 85% activity after 60 min at 0?C and 90% activity after60 min at 10?C). Among the plants examined, the most cold sensitivepyruvate,Pi dikinase was found among species of the NADP-malicenzyme subgroup. (Received March 9, 1979; )