蓖麻蚕γ-谷氨酰转肽酶的研究

L-γ-谷氨酰转肽酶(γ-GTP)广泛分布在蓖麻蚕Philosamia cynthia ricinI的中肠、马氏管、后丝腺、脂肪体、体壁等组织中;并存在于蚕个体发育中的各个阶段。酶活力以马氏管、中肠为最大。在五龄蚕前中期各组织均显示γ-GTP最大活力。在脂肪体和表皮组织中,蛹形成时又分别出现γ-GTP活力峰。昆虫生长发育必需的10种氨基酸均是蚕各组织γ-GTP酶促反应的受体。这些结果表明,γ-GTP作为L-γ-谷氨酰循环中一个关键酶,在蓖麻蚕体内对氨基酸的吸收和转运起着重要作用。     

蓖麻蚕中肠γ-谷氨酰转肽酶和底物的相互作用及其生理功能

蓖麻蚕Philosamta cynthia ricini,高度提纯的中肠γ-谷氨酰转肽酶(γ-GTP)体外转肽作用表明:L-苯丙氨酸、L-甲硫氨酸,L-半胱氨酸、L-色氨酸,L-精氨酸和L-赖氨酸是最好的γ-谷氨酰的受体,而L-谷氨酸和L-谷氨酰胺(L-Gln)系该酶良好的γ-谷氨酰供体.酶对γ-谷氨酰对硝基苯胺(γ-GNA)的K_m为0.13mmol/L(含L-苯丙氨酸)和0.29mmol/L(无L-苯丙氨酸).谷胱甘肽(GSH)和L-Gln与γ-GNA竞争酶的γ-谷氨酰结合部位,其抑制常数K₁值分别为0.5mmol/L和1.1mmol/L.Γ-GTP催化L-Gln的酰胺键水解和转肽,其催化速率相当于对γ-GNA的38%.     

蓖麻蚕中肠γ-谷氨酰转肽酶和底物的相互作用及其生理功能

蓖麻蚕Philosamta cynthia ?n,高度提纯的中肠γ-谷氨酰转肽酶(γ-GTP)体外转肽作用表明:L-苯丙氨酸、L-甲硫氨酸,L-半胱氨酸、L-色氨酸,L-精氨酸和L-赖氨酸是最好的γ-谷氨酰的受体,而L-谷氨酸和L-谷氨酰胺(L-Gln)系该酶良好的γ-谷氨酰供体。酶对γ-谷氨酰对硝基苯胺(γ-GNA)的K_m为0.13mmol/L(含L-苯丙氨酸)和0.29mmol/L(无L-苯丙氨酸)。谷胱甘肽(GSH)和L-Gln与γ-GNA竞争酶的γ-谷氨酰结合部位,其抑制常数K_1值分别为0.5mmol/L和1.1mmol/L。γ-GTP催化L-Gln的酰胺键水解和转肽,其催化速率相当于对γ-GNA的38％。     

蓖麻蚕和家蚕中的谷胱甘肽及正丁基高半胱氨酸亚砜亚胺对它的抑制作用

本文利用乙醛酸排除昆虫中比较高的游离半胱氨酸的干扰,用5,5′-二硫代双(2-硝基苯甲酸)(DTNB)方法测定了蓖麻蚕、家蚕中还原谷胱甘肽的含量及其组织分布,观察到脂肪体、后部丝腺、中肠等均含有非常丰富的谷胱甘肽(GSH),说明谷胱甘肽在昆虫氨基酸的代谢调节控制中起着重要的作用。蓖麻蚕和家蚕中,GSH含量及分布有所区别。五龄中期注射S-正丁基高半胱氨酸亚砜亚胺(BSO)引起家蚕脂肪体GSH含量明显降低,提示蚕γ-谷氨酰半胱氨酸合成酶受RSO强烈抑制;五龄后期,可能由于合成酶活力及GSH周转率均处于较低水平,未观察到GSH含量有明显下降。     

小菜蛾芳基酰胺酶表达及其与呋喃虫酰肼抗性的关系

以对硝基乙酰苯胺为底物,采用紫外分光光度计比色法,研究了小菜蛾Plutella xylostella(L.)体内芳基酰胺酶酶促反应的最适反应条件、各发育阶段和不同组织中芳基酰胺酶的活性及亚细胞分布及其与小菜蛾对呋喃虫酰肼抗性的关系。结果表明,小菜蛾芳基酰胺酶最适反应条件为35℃,pH7.5,底物浓度为1.2×10-3mol/L,反应时间30min。利用非变性聚丙烯凝胶电泳活性染色及SDS-PAGE法测得小菜蛾芳基酰胺酶的分子量为61.1ku。小菜蛾4龄幼虫的芳基酰胺酶活性最高。不同亚细胞层中,芳基酰胺酶活力大小依次为线粒体层>微粒体层>细胞核和细胞碎片层>可溶性部分。芳基酰胺酶主要分布在小菜蛾4龄幼虫的腹部,其余依次为表皮、头部和胸部。小菜蛾呋喃虫酰肼抗性品系芳基酰胺酶的活性是敏感品系的3.89倍,表明该酶参与了小菜蛾对呋喃虫酰肼的抗性形成。     

棉蚜对有机磷杀虫剂抗性的生化机理

本文对有机磷抗性和感性棉蚜Aphis gossypii三个种群抗性生化机制进行了讲究.首先用解毒酶的抑制剂测定药剂的解毒途径.进一步测定乙酰胆碱酯酶活力及其敏感性和多功能氧化酶、谷胱甘肽s-转移酶、α-乙酸萘酯酶和α-乙酸萘酯羧酸酯酶等解毒酶的活力.结果表明,体内条件下,多功能氧化酶与抗性有关,但在离体条件下,在棉蚜匀浆液中有内源抑制剂存在.α-乙酸萘酯酶和α-乙酸萘酯羧酸酯酶活力的增加,乙酰胆碱酯酶对杀虫剂敏感性的降低也是造成棉蚜对有机磷产生抗性的原因.     

β-蜕皮素对家蚕丝腺成长及合成甘氨酸、丙氨酸有关酶系的调节控制

本文研究了植源性β-蜕皮素(简称MH)对家蚕丝腺成长及合成甘氨酸、丙氨酸有关酶系的影响,探讨了5龄不同时期添食β-蜕皮素后丝腺体的成长及后部丝腺和脂肪体中丙氨酸-酮丙二酸、丙氨酸-乙醛酸和鸟氨酸转氨酶活力的变化。通过实验观察到:(1)5龄早期(饷食)添食β-蜕皮素,龄期延长8—9小时,茧层量增加,后部丝腺和脂肪体内丙氨酸-酮丙二酸转氨酶、丙氨酸-乙醛酸转氨酶在处理后6-12小时内,比对照组增高40—50%。随之酶活力略下降,至5龄后期复又升高,明显超过对照组。脂肪体中鸟氨酸转氨酶在β-蜕皮素处理后6小时和48小时均低于对照组。12—24小时以及48小时后直至老熟则明显高于对照组。(2)5龄后期(V—136小时)口腔注射β-蜕皮素,龄期缩短12—18小时。脂肪体和丝腺体中丙氨酸-酮丙二酸转氨酶、丙氨酸-乙醛酸转氨酶活力明显高于对照组(一般增高20—40%),老熟时迅速下降。本文对有关保幼激素和β-蜕皮素对5龄家蚕两种靶细胞(丝腺细胞和脂肪细胞)的相互配合和制约以完成对蚕整体的调节控制,以及这两种昆虫激素应用于蚕业生产以控制5龄期的长短和增加蚕丝产量等方面进行了讨论。     

γ-射线及大肠杆菌诱导蓖麻蚕产生抗菌物质的研究

1.用γ-射线辐照和用大肠杆菌处理蓖麻蚕五龄幼虫和蚕蛹均能诱导血淋巴产生抗菌物质,两种诱导源诱导所得活性物质的抗菌活性相似.2.研究了五龄幼虫期和蛹期诱导产生抗菌物质的动力学,发现五龄幼虫在饷食后第2—4天诱导活力最高,产生抗菌物质的持续时间亦较长;蛹期则从化蛹当天至第4天之间诱导活力较高,并可持续15天左右,高峰期一般在诱导后2天至4天之间.3.对诱导后的蓖麻蚕血淋巴进行了电泳测活和葡聚糖凝胶初步分离,发现不论幼虫期或蛹期至少可得三个活性组分,其中既有类似于P₅的大分子抗菌物质,也有类似于P_9A和P_9B的抗菌多肽;并首次发现一种分子量约70000—75000道尔顿的新抗菌蛋白.     

蓖麻蚕在变态期间代谢作用的研究——Ⅲ.酸性磷酸一酯酶的性质及其活力在变态期的变化

蓖麻蚕在变态期血淋巴酸性磷酸一酯酶活力随着蚕体的发育不断发生变化。在五龄起蚕,五龄四天及前蛹期酶活力较高,化蛹时显著降低,羽化前后又复升高。酶活力按血淋巴单位体积计算。雌雄两性差异不显著。但由于五龄中期以后,血淋巴蛋白质含量雌体大于雄体:故自五龄中期以后,酶的比活力雄体大于雌体。此酶在组织中的分布及其活力变化如下:在幼虫五龄盛食期消化道酶活力较高,其中尤以中肠酶活力最高。脂肪体和丝腺仅有微弱的酶活力。脂肪体酶活力有蛹期上升,羽化后达到最高点。此表明蓖麻蚕中肠在五龄盛食期具有旺盛的磷酸化及脱磷酸化作用,为代谢的活跃场所。在蛹及成虫体,消化道退化,脂肪体在代谢方面占了主要位置。昆虫血淋巴和其它动物血液不同的一个方面是它含有大量的磷酸酯类。影响血淋巴磷酸酯类的种类及其含量变化的,主要有两个因素:一是血淋巴本身对于不同的磷酸酸类的选择水解,蓖麻蚕在变态期血淋巴不同专一性磷酸酯酶的存在及其活力的变化,就是血淋巴选择水解的决定因素:另一是组织磷酸酯类对于血淋巴的选择分泌;本研究表明五龄幼虫中肠及脂肪体,尤其是蛹和成虫的脂肪体是血淋巴磷酸酯的重要来源。此外还研究了血淋巴磷酸一酯酶的性质,及某些金属离子,有机酸等对该酶活力的抑制及激活作用。     

β-蜕皮素对蓖麻蚕环核苷酸水平的影响

本文研究了蓖麻蚕四龄眠期及五龄幼虫期间,脂肪体、血淋巴和脑中环核苷酸水平的变化,以及注射β-蜕皮素对蓖麻蚕血淋巴、脑中环核苷酸水平的影响。发现环核苷酸水平的变化与蓖麻蚕的发育、变态有密切的关系。对蓖麻蚕五龄第五天幼虫注射β-蜕皮素(5微克/幼虫)6分钟后,血淋巴中的cGMP水平提高了将近一倍,但cAMP水平下降了23%;脑中的cGMP水平提高了4倍,cAMP水平提高了10倍。讨论了蜕皮素与环核苷酸之间的关系。     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

Absorption of toxic beta-glucosides produced by plants and their effect on tissue trehalases from insects

Trehalases present in body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor (Coleoptera), Musca domestica (Diptera), Spodoptera frugiperda and Diatraea saccharalis (Lepidoptera) were assayed in the presence and absence of toxic beta-glucosides produced by plants or their aglycones. The glucosides used were phlorizin, amygdalin, prunasin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with and without esculin. More than 60% of total trehalase activity was found in the midgut of these insects. As a rule, trehalases present in each insect were inhibited by at least two of the glucosides. Prunasin was the best inhibitor in tissues with highest trehalase activity. S. frugiperda beta-glucosidases were not able to hydrolyze esculin. Nevertheless, their larval midguts absorb the intact glucoside that is recovered from the fat body, Malpighian tubules and mainly from haemolymph. Mature larvae fed on a diet containing 3 mM (0.1%) esculin have 0.2 mM esculin in their haemolymph, and weigh 60% of control larvae. In vitro, haemolymph trehalase activity is abolished by 0.5 mM esculin. This inhibition may play a role in the decrease of body weight and in animal survival. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activity in tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why the S. frugiperda development is not impaired by 1% dietary amygdalin, in contrast to what is observed when insects are reared in 0.1% esculin. The data suggest that many plant beta-glucosides are toxic because they inhibit trehalase, a key enzyme controlling glucose availability in insects.     

Age- and diapause-related acid and alkaline phosphatase activities in the intestine and malpighian tubules of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

Specific activities for soluble (s) and membrane (m)-bound acid (ACP) and alkaline phosphatases (ALP) were determined in the midgut, hindgut, and Malpighian tubules for developing, prediapausing, and diapausing adult Colorado potato beetles, Leptinotarsa decemlineata (Say). High ACP activities were found in the hindgut and Malpighian tubules while high ALP activities were found in the Malpighian tubules. Variation in both ACP and ALP activities in each tissue reflects fluctuation in protein synthesis and secretion involved with digestion, excretion, and other unknown functions. Phosphatase activities in the tissues examined show the dynamic nature of diapause in this insect. Diapausing beetles showed increases in phosphatase activity after hormone treatments. JHA treatments increased s-ACP and m-ACP activities in all tissues but 20-HE did not increase activity in any tissue. Allatotropin tended to mimic the effects of JHA treatment. The s-ALP activity was also increased in all tissues whereas m-ALP was increased in the midgut and hindgut by JHA treatment. Malpighian tubule m-ALP activity was only increased by 20-HE treatments. Allatotropin was not as effective in increasing ALP activities as it was with ACP activities.     

Expression patterns of genes encoding proteins with peritrophin A domains and protein localization in Mamestra configurata

Genes encoding three proteins (McPPAD1-3) with peritrophin A chitin-binding domains (PADs) were identified from a Mamestra configurata larval midgut cDNA library. In addition to midgut, McPPAD1-3 and a previously identified gene encoding the peritrophin, McPM1, were expressed in foregut, hindgut, Malpighian tubules, tracheae, fat body and cuticle; however, the corresponding McPPAD proteins exhibited different localization patterns. McPPAD1 was restricted to the digestive tract and Malpighian tubules, McPPAD2 to Malpighian tubules, and McPPAD3 to the foregut, midgut, hindgut, tracheae and cuticle. Protein fold recognition analysis using tachycitin as a guide structure modelled the McPPAD1 PADs, but not McPPAD2 or McPPAD3 PADs. The McPPAD1 PADs were predicted to contain three anti-parallel β-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.     

An overview of gene activity in the fat body of Drosophila gibberosa

In the larval fat body of Drosophila gibberosa, polytene chromosome structure and activity exhibit cytological differences from chromosomes of midgut and salivary glands. These differences include long-persisting puffs, transient puffs and long-persisting band modulations. Some early ecdysteroid-induced puffs are present in all three organs but few late puffs are present in the fat body. Comparative studies reveal, therefore, that late larval-early pupal puffing is enhanced in salivary glands relative to gut, fat body and Malpighian tubules. After the fat body breaks up in the prepupa, the rate of programmed cell death and the corresponding slow decline of chromosomal activity also differ from cell to cell and from other organs.by M.L. Pardue     

Excretory role of the midgut in larvae of the tobacco hornworm, Manduca sexta (L.).

Caterpillars of Manduca sexta use two distinct transport mechanisms for the excretion of dyes. One pump (Type A) has a high affinity for acid (anionic) dyes and occurs in the midgut and medial Malpighian tubules. Acid dyes accumulate rapidly in the lumen of the midgut while the Malpighian tubules appear to play only a minor role in the excretion of these dyes. The other pump (Type B) excretes basic (cationic) dyes and is located primarily in the proximal Malpighian tubules. Evidence is presented that hippuric acid competes with acid dyes for excretion by both midgut and Malpighian tubules. After the final-instar larva purges its gut the ability of the midgut and Malpighian tubules to excrete dyes gradually decreases. Sixty hours after the purge only the Malpighian tubules retain some dye excreting activity.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

The influence of ouabain on the elimination of injected and orally applied dyes in Drosophila hydei

The sour dyes azocarmine and indigocarmine are excreted through the Malpighian tubules and the midgut after injection into the body cavity of third instar Drosophila hydei larvae. After injection, the other organs are free of dyes. The epithelium of the midgut does not allow orally applied dyes to pass into the haemolymph. Ouabain diminishes significantly the content of dyes in the cavity of the Malpighian tubules and of the midgut. The maximal concentration of azocarmine decreases in the Malpighian tubules to about 65 per cent and in the midgut to about 70 per cent. Indigocarmine decreases in the Malpighian tubules to about 55 per cent. The content of indigocarmine of the midgut does not change significantly after ouabain injections. As ouabain inhibits active ion transport, the decrease of the concentration of dyes is seen as proof of the coupling of active ion transport processes and of excretion of the dyes. Moreover, this decrease points to an ouabain-sensitive transport mechanism, which is localized in the epithelia of the Malpighian tubules and midgut.     

Ultrastructural changes in the gut and the Malpighian tubules of Epilachna varivestis after application of the new antibiotic Nikkomycin involve an osmoregulatory defect

The effect of the antibiotic Nikkomycin was investigated on the Malpighian tubules and the gut of fourth-instar larvae of the Mexican bean beetle, Epilachna varivestis. Within the Malpighian tubules, three different stages in cell alterations can be recognized. A stage of increased activity (Stage A), and two stages of dedifferentiation (Stages B and C) which are distinguishible by characteristic mitochondrial morphology. In Stage C individuals, when Malpighian tubule function stops entirely, alterations in the midgut take place, that are signs of increased activity. Measurements of hemolymph osmotic pressure showed that there is a considerable increase to a higher level which is maintained. Compared with the ultrastructural data, the regulation of osmotic pressure on a higher level may, in part, be the result of compensation for the failure of Malpighian tubule function by the midgut.