大豆胰蛋白酶抑制剂对棉铃虫幼虫消化生理和生长发育的影响

本研究根据棉铃虫Helicotverpa ormigera(Hubner)幼虫中肠蛋白酶在离体条件下对蛋白酶抑制剂的反应,选择具有较强抑制作用的大豆胰蛋白酶抑制剂,以0.21-4.2%(干重)的浓度配入幼虫人工饲料,测定了幼虫短期和长期取食这些饲料引起的中肠类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力的变化和生长抑制效应。短期取食抑制剂的幼虫,中肠弱碱性类胰蛋白酶活力显著增高,在4.2%。浓度下比对照高出21%;强碱性类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力显著降低,生长发育受到明显抑制。长期取食低浓度(0.84%)抑制剂的幼虫,弱碱性类胰蛋白酶和类胰凝乳蛋白酶活力显著增高,强碱性类胰蛋白酶活力显著降低。总蛋白酶活力变化不显著;长期取食高浓度(4.2%)抑制剂的幼虫,强碱性类胰蛋白酶和总蛋白酶活力显著降低,其它酶活力变化不显著。抑制剂随浓度的增高对幼虫生长的抑制作用加强,但浓度高于0.84%后,抑制强度的变化减小。据此作者认为,蛋白酶抑制剂对昆虫抗营养效应在于它对蛋白酶的激活和抑制作用,从而导致各种蛋白酶间的协调性破坏,昆虫消化过程受阻,影响生长发育。     

pH对红褐斑腿蝗中肠主要蛋白酶活性的影响

为评价pH对红褐斑腿蝗Catantops pinguis (Stål)中肠蛋白酶活性的影响, 本文用3种专性底物测定了不同pH环境下蝗虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性。结果表明: 雄性红褐斑腿蝗中肠肠液的pH值为6.92±0.043, 雌性为7.03±0.054, 两性间差异不显著（P>0.05）。并且发现3种蛋白酶的最适pH值各不相同, 其中雌雄虫的强碱性类胰蛋白酶（以BAPNA为底物）最适pH分别为8.5和10.5; 雌雄虫的弱碱性类胰蛋白酶（以TAME为底物）最适pH分别为9.0和9.5; 而雌雄虫的类胰凝乳蛋白酶（以BTEE为底物）最适pH雌性为8.5, 雄性为8.0。统计结果显示, pH对红褐斑腿蝗中肠蛋白酶活性影响显著（P<0.01）, 两性间蛋白酶活性差异显著（P<0.01）。在最适pH情况下, 雌性的类胰蛋白酶活性高于雄性, 而类胰凝乳蛋白酶活性则是雄性高于雌性。在中肠pH范围内雌性比雄性具有更高的消化蛋白酶活性, 显示雌性具有较强的食物处理能力以摄取更多的营养物质为繁殖活动（孕卵）作准备, 而该种蝗虫最适pH范围较宽, 可能与其取食植物范围较宽有关。     

胰蛋白酶与底物在尿素溶液中的结合

胰蛋白酶的专一性底物,对甲苯磺酰-L-精氨酸甲酯,对酶在236mμ的尿素差吸收峰有熄灭作用;不同量的对甲苯磺酰-L-精氨酸甲酯对胰蛋白酶236mμ的尿素差吸收值的影响形成一滴定曲线。在8M尿素溶液中对甲苯磺酰-L-精氨酸甲酯和胰蛋白酶的旋光不具有加和性。这些事实说明在尿素溶液中胰蛋白酶能够和底物结合。二异丙磷酰胰蛋白酶的236mμ尿素差吸收峰亦能被对甲苯磺酰-L-精氨酸甲酯熄灭,指出二异丙磷酰胰蛋白酶仍然具有和对甲苯磺酰-L-精氨酸甲酯结合的能力。用N-溴代琥珀酰亚胺氧化胰蛋白酶的色氨酸残基后,酶的尿素差示光谱消失以及酶的pH差示光谱中反映了一个pK2.8的解离基团,说明色氨酸残基和羧基可能直接或间接与236mμ左右的差示光谱有关。从底物存在下胰蛋白酶在pH1.6—3.6范围内不再呈现pH差示光谱看来,羧基可能参与底物的结合。     

慈菇蛋白酶抑制剂通过花粉管途径对小麦的导入及转基因植株分析

慈菇蛋白酶制剂（ＡｒｒｏｗｈｅａｄＰｒｏｔｅｉｎａｓＩｎｈｉｂｉｔｏｒ,ＡＰＩ）是来源于慈菇（Ｓａｇｉｔｔａｒｉａｔｒｉｆｏｌｉａ）储藏器官的一种天抗虫物质,属于丝氨酸蛋白酶抑制剂类,能抑制胰蛋白酶、胰凝乳蛋白酶和激肽释放酶,对某些鳞翅目,双翅目以及鞘翅目等昆虫有毒杀作用。     

蛋白酶抑制剂对梨小食心虫幼虫中肠蛋白酶活性的影响

【目的】梨小食心虫Grapholitha molesta(Busck)是一种危害极其严重的果树害虫。中肠蛋白酶在昆虫生长发育过程中起着重要作用。本研究测定梨小食心虫幼虫中肠内蛋白酶活性的最适p H、蛋白酶抑制剂和激活剂对蛋白酶活性的作用,为利用蛋白酶抑制剂防治该害虫提供新思路。【方法】提取梨小食心虫3龄幼虫中肠液,利用酶专性底物测定各蛋白酶在3种不同缓冲溶液中的最适p H(dd H2O为对照)、蛋白酶抑制剂和激活剂对中肠蛋白酶活性的影响,同时测定饲喂蛋白酶抑制剂(PMSF,TLCK,TPCL和STI)后梨小食心虫中肠蛋白酶活性的变化。【结果】梨小食心虫幼虫中肠总蛋白酶在Tris-HCl,KH2PO4/Na OH和Glycine/Na OH 3种缓冲液中最适p H分别为10.5,11.0和11.0,强碱性胰蛋白酶的最适p H分别为10.5,11.0和11.0,弱碱性胰蛋白酶的最适p H分别为8.5,9.0和9.0,胰凝乳蛋白酶的最适p H分别为8.5,9.0和9.5。5种蛋白酶抑制剂(DTT,PMSF,TLCK,TPCL和STI)中,除TLCK对凝乳蛋白酶激活外,其他蛋白酶抑制剂对4种蛋白酶均表现为抑制,且浓度越大抑制效应越明显。抑制剂DTT对总蛋白酶和弱碱性胰蛋白酶的抑制效果高于其他抑制剂。4种蛋白酶激活剂(Mg Cl2,Ca Cl2,EDTA和EGTA)中,Mg Cl2抑制总蛋白酶和胰凝乳蛋白酶活性,而激活胰蛋白酶活性;Ca Cl2激活总蛋白酶和弱碱性胰蛋白酶活性,而抑制强碱性胰蛋白酶和胰凝乳蛋白酶,EDTA对4种蛋白酶均表现为抑制,EGTA除对强碱性胰蛋白酶表现为激活外,对另外3种蛋白酶表现抑制。用蛋白酶抑制剂PMSF,TLCK,TPCL和STI饲喂梨小食心虫幼虫,各抑制剂均可抑制4种蛋白酶活性,且在不同取样时间抑制水平不同。其中STI(50μg/m L)对4种蛋白酶的抑制效果高于其他抑制剂,且浓度越大抑制效应越明显。10,20和50μg/m L STI 3种浓度处理组,在取食后4 h时,4种蛋白酶活性升高,且上升程度与STI浓度有关;酶活性在20μg/m L STI处理后48 h,50μg/m L STI处理后60 h时最低,抑制剂STI表现出持效性。【结论】蛋白酶抑制剂对梨小食心虫幼虫中肠蛋白消化酶的活性具有一定的抑制作用,其中大豆胰蛋白酶抑制剂STI在害虫防治中具有极其重要的应用价值。     

三种鳞翅目幼虫肠道蛋白水解酶的活性

本工作以酪蛋白为底物,测定粘虫Mythimna separata Walker、棉铃虫Heliothis armigera Hubner和大蜡螟 Galleria mellonlla Linnaeus三种鳞翅目幼虫肠道蛋白水解酶的活性,并分别用BTEE和TAME为底物,测定了其中类胰凝乳蛋白酶和类胰蛋白酶的活性.结果表明:三种幼虫肠道都含有类胰凝乳蛋白酶和类胰蛋白酶.抑制剂TPCK可以部分地抑制类胰凝乳蛋白酶的活性,而胰酶抑制剂则显著地抑制类胰蛋白酶的活性.     

五种寄主植物对甜菜夜蛾幼虫中肠蛋白酶活性的影响

甜菜夜蛾Spodoptera exigua(Hübner)是重要的多食性、暴食性农业害虫之一。其寄主广泛,可危害禾谷类、甜菜、蔬菜、棉花等作物及杂草。昆虫中肠胰蛋白酶和胰凝乳蛋白酶在昆虫消化和生长发育中起重要的作用。本研究分别用辣椒、豇豆、玉米、甘蓝、反枝苋5种寄主植物饲喂甜菜夜蛾幼虫,利用酶专性底物分别测定了甜菜夜蛾幼虫中肠总蛋白酶活性、强碱性胰蛋白酶活性、弱碱性胰蛋白酶活性和胰凝乳蛋白酶活性。结果表明:甜菜夜蛾中肠总蛋白酶活性、强碱性胰蛋白酶活性、弱碱性胰蛋白酶活性以及胰凝乳蛋白酶活性在不同寄主植物之间存在显著差异,取食反枝苋甜菜夜蛾各中肠蛋白酶活性均高,取食甘蓝的各中肠蛋白酶活性均低,而取食玉米、豇豆、辣椒居中且活性差异不显著。取食同一寄主植物(反枝苋或甘蓝)不同龄期甜菜夜蛾幼虫的中肠蛋白酶活性也各不相同,随着龄期的增加,各中肠蛋白酶活性总体呈下降趋势,2龄幼虫的酶活性偏高,而5龄的酶活性显著低于其他龄期的酶活性。本研究明确了不同寄主植物对甜菜夜蛾幼虫中肠蛋白酶活性的影响,结果对明确甜菜夜蛾对寄主的适应性及对以中肠蛋白酶为靶标的害虫防治具有一定参考价值。     

The effects of Cry2Ab and Cry1Ac insecticidal proteins on protease activity in the midgut of Helicoverpa armigera

为明确Cry2Ab和Cry1Ac 2种Bt杀虫蛋白单用与混用对棉铃虫Helicoverpa armigera(Hübner)中肠主要蛋白酶活性的影响,本文测定了取食含不同Bt蛋白人工饲料后棉铃虫中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的差异.结果发现:Cry2Ab处理12h后对棉铃虫中肠总蛋白酶影响不大;对类胰蛋白酶的影响最大,除最高浓度处理外,其他浓度处理后棉铃虫类胰蛋白酶的活性明显高于对照;但对类胰凝乳蛋白酶活性的影响呈倒“V”字型,只有6.67μg／g Cry2Ab处理后的棉铃虫酶活力显著高于对照,其他浓度处理与对照差异不显著或略低于对照;随着取食含Cry2Ab饲料时间的增加,棉铃虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性比对照显著增加;与对照相比,处理36h后类胰蛋白酶活性最高可增加到6.43倍.Cry1Ac处理棉铃虫12h后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性都明显增加,而且与处理浓度呈正相关;但是24h后,处理后棉铃虫的总蛋白酶和类胰凝乳蛋白酶活性明显降低,只有类胰蛋白酶活性仍高于对照,但活性增长倍数低于12h时的处理.Cry2Ab和Cry1Ac 2种蛋白混用处理棉铃虫后,2种酶的酶活力基本低于Cry1Ac和Cry2Ab单用的酶活力之和;只有2种蛋白浓度均为2.22μg/g混用时,处理12b后类胰蛋白酶和类胰凝乳蛋白酶的活性高于2种蛋白单用时酶活力之和,且都显著的高于对照.     

Cry2Ab及Cry1Ac杀虫蛋白对棉铃虫中肠蛋白酶活性的影响

为明确Cry2Ab和Cry1Ac2种Bt杀虫蛋白单用与混用对棉铃虫Helicoverpa armigera（Htibner）中肠主要蛋白酶活性的影响,本文测定了取食含不同Bt蛋白人工饲料后棉铃虫中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的差异。结果发现：Cry2Ab处理12h后对棉铃虫中肠总蛋白酶影响不大;对类胰蛋白酶的影响最大,除最高浓度处理外,其他浓度处理后棉铃虫类胰蛋白酶的活性明显高于对照;但对类胰凝乳蛋白酶活性的影响呈倒“V”字型,只有6．67ug／gCry2Ab处理后的棉铃虫酶活力显著高于对照,其他浓度处理与对照差异不显著或略低于对照;随着取食含Cry2Ab饲料时间的增加,棉铃虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性比对照显著增加;与对照相比,处理36h后类胰蛋白酶活性最高可增加到6．43倍。Cry1Ac处理棉铃虫12h后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性都明显增加,而且与处理浓度呈正相关;但是24h后,处理后棉铃虫的总蛋白酶和类胰凝乳蛋白酶活性明显降低,只有类胰蛋白酶活性仍高于对照,但活性增长倍数低于12h时的处理。Cru2Ab和Cry1Ac2种蛋白混用处理棉铃虫后,2种酶的酶活力基本低于Cry1Ac和Cry2Ab单用的酶活力之和;只有2种蛋白浓度均为2．22ug／g混用时,处理12h后类胰蛋白酶和类胰凝乳蛋白酶的活性高于2种蛋白单用时酶活力之和,且都显著的高于对照。     

棉铃虫中肠类胰蛋白酶的部分纯化和性质测定及其对苏云金杆菌δ—内毒素的降解作用

将棉铃虫Helicoverpa armingera中肠液经SephadexG-75、DEAE-SephadexA-50阴离子交换和CM-SephadexC-50阳离子交换柱层析进行分离纯化,得到部分纯化的24.5kD蛋白,该蛋白可以水解胰蛋白酶的专性底物BApNA和TAME,不能水解胰凝乳蛋白酶的专性底物BTEE,蛋白酶抑制剂的抑制试验显示,TLCK、PMSF、STI及SBBI均可显著抑制该蛋白对BApNA的水解作用,而TPCK无抑制作用,由此推断,24.5kD蛋白是棉铃虫的类胰蛋白酶。以BApNA为底物,类胰蛋白酶的最适pH为10.5-11.0。SephadexG-75分离得到了4个洗脱峰,电泳结果显示,各洗脱峰均能使苏云金杆菌库斯塔克变种Bacillus thuringiensis var.kurstakiHD-1原毒素降解为60.5kD的活力片段,峰Ⅳ虽然不能水解BApNA和TAME,但仍可对原毒素进行降解,而类胰蛋白酶活力最高的峰Ⅱ已将60.5kD蛋白完全降解,当减少用量至1/3时,才出现60.5kD蛋白。因此,苏云金杆菌δ-内毒素在棉铃虫中肠内的降解有多种蛋白酶参与,类胰蛋白酶在其中起重要作用,CM-SephadexC-50进一步分离得到的类胰蛋白酶可对原毒进行降解,其降解作用受到TLCK、PMSF、STI及SBBI的显著抑制,不受TPCK的影响。     

Genetic Engineering Techniques: Recent Developments : Edited by P.C. Huang,T.T. Kuo and R. Wu Academic Press; New York, 1982 360 pages. $28.50

Extracts from white croaker skeletal muscle showed two alkaline proteases and a trypsin inhibitor when they were chromatographed in DEAE-Sephacel. The activity against azocasein was maximal at pH 8.5 and 9.1 for proteases I and II, respectively. Both enzymes showed optimum activity at 60° C. The molecular masses were found to be 132 kDa for protease 1,363 kDa for protease II, and 65 kDa for the inhibitor. Protease I showed the characteristics of a trypsin-like enzyme, and protease II those of a SH-enzyme. These proteins may play important roles in mechanisms of cellular proteolysis.     

Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.     

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.     

Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.     

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.     

转Bt基因抗虫玉米对亚洲玉米螟幼虫几种主要酶系活性的影响

研究了表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米对亚洲玉米螟Ostrinia furnacalis (uenée) 幼虫解毒酶、保护酶和中肠蛋白酶活性的影响,测定比较了取食转Bt基因玉米后幼虫体内α-乙酸萘酯酶、乙酰胆碱酯酶、谷胱甘肽S-转移酶、过氧化氢酶、超氧化物歧化酶、中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶的活力。结果表明,取食转Bt基因玉米48 h后亚洲玉米螟幼虫体内的α-乙酸萘酯酶、谷胱甘肽S转移酶活力明显低于对照;而乙酰胆碱酯酶活力显著高于对照,在取食48 h、60 h和72 h的活力分别是对照的2.00、1.50和2.50倍。保护酶系、中肠总蛋白酶、弱碱性类胰蛋白酶和类胰凝乳蛋白酶的活性在取食48 h后明显受到抑制;但强碱性类胰蛋白酶的活性显著高于对照,取食48 h、60 h和72 h的活力分别是对照的4.00、1.67和1.33倍。乙酰胆碱酯酶和强碱性类胰蛋白酶可能与亚洲玉米螟对Bt的抗性有关。     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris

Based on substrate specificity, an alkaline pH optimum, sensitivity to selected proteinase inhibitors, and molecular analysis, we provide evidence for the presence of a trypsin-like serine proteinase in the salivary gland complex (SGC) of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The predominant activity in extracts of the SGC against N(2)-benzoyl-L-arginine-p-nitroanilide (L-BApNA) was at pH 10, but a minor peak of activity also occurred at pH 5. The major BApNAase activity focused at 10.4 during preparative isoelectric focusing and was eluted with an apparent molecular weight of 23,000 from a calibrated gel filtration column. The BApNAase fraction gave a single major band when analyzed on a casein zymogram. The activity was completely suppressed by the serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor. A cDNA coding for a trypsin-like protein in the salivary glands of L. lineolaris was cloned and sequenced. The 971bp cDNA contained an 873-nucleotide open reading frame encoding a 291-amino acid trypsin precursor. The encoded protein included amino acid sequence motifs that are conserved with four homologous serine proteases from other insects. Typical features of the putative trypsin-like protein from L. lineolaris included the serine protease active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, the residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for trypsin like enzymes in the salivary glands of L. lineolaris.     

12a-羟基鱼藤酮对斜纹夜蛾生殖力的影响及其作用机理

为探讨12a-羟基鱼藤酮对斜纹夜蛾Prodenia litura (Fabricius)生殖力的影响及其作用机理, 本文测定了该虫取食12a-羟基鱼藤酮后的产卵量。在用高效液相色谱仪确定其卵巢组织中存在12a-羟基鱼藤酮后, 通过MTT法测定该化合物对斜纹夜蛾卵巢细胞（PL细胞）的细胞毒性,并利用流式细胞仪检测该化合物对PL细胞细胞周期、膜电位、胞内钙离子浓度、线粒体膜电位的影响。结果表明:斜纹夜蛾幼虫取食12a-羟基鱼藤酮后产卵量下降80％以上, 并从卵巢组织中检测出该化合物。12a-羟基鱼藤酮对PL细胞增殖具有抑制活性, 其IC₅₀为6.6 mg/L。该化合物将PL细胞周期阻滞于S期, 导致PL细胞膜电位、线粒体膜电位和细胞内游离钙离子浓度均显著升高。该化合物导致斜纹夜蛾繁殖力降低。12a-羟基鱼藤酮将PL细胞增殖阻滞于细胞周期的S期。同时, 该化合物对卵巢细胞具有较弱的毒杀活性, 导致一些卵巢细胞死亡。由于上述原因, 卵巢细胞数量逐渐减少而导致卵巢管萎缩。卵巢管的萎缩使卵巢管内的卵母细胞不能发育成卵细胞, 故其生殖力下降。