Hydrolysis of phospholipids by a lysosomal enzyme

The phospholipid-hydrolyzing activity of rat liver lysosomes has been studied. These lysosomes contain a phospholipase that cleaves both fatty acid ester linkages of lecithin and of phosphatidyl ethanolamine and releases free fatty acids from both positional isomers of lysolecithin. The enzyme does not require calcium for maximum activity, and is inhibited by diethyl ether and sodium deoxycholate. Mercuric ions and cetyltrimethyl ammonium bromide also inhibit the hydrolysis. Compared with lipase activity, this enzyme is relatively stable to heat. The specific activity of the hydrolysis of lecithin by the lysosomal enzyme is considerably higher than those reported for mitochondrial and microsomal phospholipases. The enzyme resembles other hydrolases of the lysosome in that it has an acid pH optimum (pH 4.5). This enzymic activity is present in both the lysosomal soluble enzyme fraction and in the lysosomal membrane fraction. The enzyme may participate in the intracellular digestion of mitochondria that is carried out by the intact lysosome in vivo. Localized inflammation and changes in vascular permeability following tissue damage could be catalyzed by this phospholipase.     

棉铃虫不同虫态及虫龄血淋巴中酚氧化酶活力的比较

分别测定了棉铃虫Helicoverpa armigeraHübner不同虫态及虫龄血清和血细胞中酚氧化酶(phenoloxidase,PO)的活力。结果显示血清和血细胞中都有酚氧化酶活性,且血细胞中高于血清中。不同虫态及虫龄的血清和血细胞中酚氧化酶活力有很大的不同,血清和血细胞中酚氧化酶活力变化规律一致。3龄幼虫酶活力最高,5龄幼虫最低。酶活力大小依次为:3龄幼虫>预蛹>4龄幼虫>蛹>5龄幼虫     

Influence of the braconid Glyptapanteles liparidis on the juvenile hormone titer of its larval host,the gypsy moth,Lymantria dispar

The increase in the juvenile hormone (JH) III titer in the hemolymph of Lymantria dispar larvae that were parasitized by the endoparasitoid braconid, Glyptapanteles liparidis, during the host's premolt to third instar, coincided with the molt of the parasitoid larvae to the second instar between day 5 and 7 of the fourth host instar. It reached a maximum mean value of 89 pmol/ml on day 7 of the fifth instar while it remained below 1 pmol/ml in unparasitized larvae. Only newly molted fifth instar hosts showed a low JH III titer similar to that of the unparasitized larvae. JH II, which is the predominant JH homologue in unparasitized gypsy moth larvae, also increased relative to controls in the last two samples (days 7 and 9) from parasitized fourth and fifth instars. Compared to unparasitized larvae, a generally reduced activity of JH esterase (JHE) was found in parasitized larvae throughout both larval stages. The reduction in enzyme activity at the beginning and at the end of each instar, when the JHE activity in unparasitized larvae was high, may be in part responsible for the increased JH II and JH III titers in parasitized larvae. Ester hydrolysis was the only pathway of JH metabolism in the hemolymph of unparasitized and parasitized gypsy moth larvae as detected by chromatographic assays. © 1996 Wiley-Liss, Inc.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.     

粘虫谷氨酰胺转胺酶(MsTGase)活力测定条件的正交优化及其在幼虫体内的分布

【目的】本研究旨在优化Grossowicz氧肟酸比色法测定粘虫Mythimna separata谷氨酰胺转胺酶(Ms TGase)活力的组合条件,以Ms TGase酶活力为依据分析其在不同龄期幼虫体内的分布规律。【方法】取4龄粘虫幼虫,通过组织匀浆和沉析纯化制备Ms TGase,采用Grossowicz比色法测定Ms TGase酶活力,并对Grossowicz比色法的多重实验因素进行正交优化,进一步结合差速离心法分析不同龄期幼虫体内和亚细胞组分(细胞核和细胞碎片,线粒体,微粒体以及胞质溶胶)中Ms TGase酶活力。【结果】结果表明,酶浓度、底物浓度、反应体系pH值、反应温度及钙离子浓度等实验因素都对Ms TGase酶活力测定结果产生显著影响,其影响大小顺序为:酶浓度>温度> p H>底物浓度>Ca2+浓度。Ms TGase酶比活力测定的最优化条件:酶浓度20 mg/m L、底物浓度0. 04 mol/L、反应体系pH值6. 5、测定温度37℃,不添加钙离子。在1-5龄幼虫中以4龄幼虫的Ms TGase酶活力最高,其比活力也显著高于其他龄期的,且在1-5龄幼虫胞质溶胶中Ms TGase酶活力分别占各亚细胞组分酶活力总和的39%,25%,48%,60%和61%。【结论】所获得的最优化条件适用于粘虫Ms TGase酶活力测定。Ms TGase在粘虫体内呈显著的龄期表达特征和亚细胞分布规律。     

马尾松毛虫过氧化氢酶及过氧化物酶与耐药性的关系

马尾松毛虫Dendrolimus punctatus幼虫体内存在着过氧化氢酶(CAT)和过氧化物酶(POD)。4龄幼虫的CAT和POD活力较大,其次是6龄幼虫,5龄幼虫的CAT和POD活力较4龄和6龄幼虫低。醚菊酯(etofenprox)处理后,在兴奋期(30 min),CAT和POD活力水平上升。4龄和6龄幼虫在抑制期(50 min以后),CAT和POD活力呈波动式上升,接近死亡时下降。5龄幼虫的CAT和POD活力呈波动式上升,接近死亡时下降。5龄幼虫的CAT在抑制期保持比正常虫体高的活力。结果表明,马尾松毛虫4龄、5龄和6龄幼虫与耐药性存在一定的相关性,研制酶的抑制剂具有实用意义。根据毒力测定结果,马尾松毛虫幼虫对醚菊酯的耐药力,5龄是4龄的1.43倍,6龄是4龄的1.72倍。因此,药物防治的合理时期应掌握在4龄以前较适宜。     

转cry1Ab/cry1Ac基因水稻对大螟幼虫体内三种保护酶活性的影响

为阐明转cry1Ab/cry1Ac基因水稻对大螟Sesamia inferens (Walker)作用的生理生化机制, 本研究用转cry1Ab/cry1Ac基因水稻茎秆饲喂大螟3龄和5龄幼虫, 采用酶活性测定方法研究了取食转Bt水稻对大螟幼虫体内3种保护酶SOD(superoxide dismutase)、 CAT(catalase)和POD(peroxidase)活性的影响。结果表明, 大螟3龄幼虫在取食转基因水稻24 h后SOD活性与对照相比提高了43.44%, 48 h后降至最低值; 在取食24 h后POD值达到最高值, 其酶活性比对照升高了29.22%, 最终在取食48 h后降至最低值, 并显著低于对照; 在取食转基因水稻4 h后, CAT活性升高了30.33%, 在取食48 h后, 与对照相比, CAT活性降低了27.01%; 5龄幼虫取食4 h后SOD活性显著高于对照水平, 36 h后降至最低值, 与对照相比, 活性下降了31.62%; 在取食8 h后POD活性达到最高值, 与对照相比, 升高了73.20%, 36 h后酶活性降至最低值; 在取食之初4 h CAT活性达到最高值, 与对照相比, 其值升高了75.73%, 在取食48 h后, 其活性与对照相比减少了7.55%。3龄幼虫与5龄幼虫相比, 对Bt的抗性水平较低, 自身防卫能力较差。结果说明, 在取食初期, 试虫体内保护酶活性升高, 以抵御Bt毒蛋白对虫体的伤害作用, 随着取食时间的延长, 保护酶活性迅速降低, 从而干扰虫体正常的代谢过程, 导致虫体出现中毒症状, 致使昆虫死亡。     

氟铃脲对斜纹夜蛾酚氧化酶活性的影响

【目的】探讨氟铃脲对斜纹夜蛾Spodoptera litura酚氧化酶活性的影响.【方法】应用酶动力学法,在确定斜纹夜蛾酚氧化酶最适反应条件基础上,测定氟铃脲与酚氧化酶直接作用及氟铃脲处理幼虫后酚氧化酶的活性.【结果】以邻苯二酚为底物,斜纹夜蛾酚氧化酶的最适pH 6.5,最适温度30℃.氟铃脲没有和酚氧化酶结构中的铜离子结合,而是直接与酶蛋白发生作用.氟铃脲浓度低于1 153 mg/L对离体酚氧化酶有激活作用,在138 mg/L激活作用最大,浓度高于1 153 mg/L则产生抑制作用.用46 mg/L和92 mg/L亚致死剂量氟铃脲连续处理5龄幼虫不同时间后,酚氧化酶活性均显著提高,且高浓度的激活作用大于低浓度的激活作用.进一步测定药剂处理幼虫24,48和72 h的血淋巴、表皮和头部酚氧化酶活性,随着氟铃脲处理时间延长,两个剂量的氟铃脲对血淋巴、表皮及头部酚氧化酶的激活作用逐渐增大,氟铃脲对血淋巴酚氧化酶的激活作用最大,表皮次之,头部最低.氟铃脲处理幼虫后,其预蛹和蛹酚氧化酶的活性也显著提高,表现出一定的后效应.【结论】一定浓度范围内,氟铃脲对斜纹夜蛾酚氧化酶具有激活作用.     

Cyclic AMP phosphodiesterase activity in the midgut of Manduca sexta

Midgut preparations from Manduca sexta larvae exhibit potent cyclic AMP phosphodiesterase activity. The enzyme exhibits a pH optimum at pH 8·8 and the assay reaction is linear for at least 30 min. Enzyme activity is greatest in larvae during the second day of the fourth and fifth instars and decreases at the end of the instar. Midgut cyclic AMP phosphodiesterase activity rises again in the pharate pupa and during pharate adult development. Whether these alterations in emzyme activity reflect humorally controlled morphogenetic changes in the midgut or are involved in the physiological functions of the midgut is as yet not known.     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

Cloning, expression, and localization of a molt-related beta-N-acetylglucosaminidase in the spruce budworm, Choristoneura fumiferana

A beta-N-acetylglucosaminidase cDNA (CfGlcNAcase) was cloned from the spruce budworm, Choristoneura fumiferana. Western blotting analysis of developmental CfGlcNAcase expression revealed high levels of expression of the gene on the last day of the 5th instar larvae and the first day in the 6th instar larvae, followed by a decrease to background levels during the intermolt of the 6th instar. CfGlcNAcase was detected again from the last day of the 6th instar to day 2 of pupal stage. CfGlcNAcase expression was induced by tebufenozide at 24 h post treatment and remained at high levels until 72 h. Immunohistochemical localization analysis of CfGlcNAcase indicated that CfGlcNAcase was present in the molting fluid, epidermis, trachea, and hemolymph in prepupae during the transformation from larva to pupa. CfGlcNAcase cDNA was expressed into a recombinant protein in bacterial and baculovirus systems and the protein expressed in the baculovirus system had a higher chitinolytic activity than in the bacterial system and appeared to be secreted.     

Activity of gut alkaline phosphatase,proteases and esterase in relation to diapause of pharate first instar larvae of the gypsy moth,Lymantria dispar

Two distinctly different patterns of gut enzyme activity were noted in relation to diapause in pharate first instar larvae of the gypsy moth, Lymantria dispar. Trypsin, chymotrypsin, elastase, aminopeptidase and esterase activities were low at the initiation of diapause and through the period of chilling needed to terminate diapause. At the completion of a 150 day chilling period, activity of each of these enzymes quickly increased when the pharate larvae were transferred to 25°C. By contrast, activity of alkaline phosphatase (ALP) increased rapidly at the onset of diapause, remained elevated throughout diapause, increased again during postdiapause, and then dropped at the time of hatching. In addition, zymogram patterns of ALP activity differed qualitatively in relation to diapause: several bands were detectable during the pre- and postdiapause periods, but only one band, a band of high mobility, was visible during diapause. The ALP isozyme present in diapausing pharate larvae had a pH optimum of 10.6. Diapause in the gypsy moth can be averted by application of an imidazole derivative, KK-42, and pharate larvae treated with KK-42 showed elevated protease and esterase activity, low ALP activity, and expressed ALP isozymes with low mobility. Thus the overall patterns of gut enzyme activity and the ALP zymogram in KK-42 treated individuals were similar to those observed in untreated individuals at the termination of diapause. Our results suggest a unique pattern of enzyme activity in the gut that is regulated by the diapause program. Arch. Insect Biochem. Physiol. 37:197–205, 1998. © 1998 Wiley-Liss, Inc.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Parasitism of Lacanobia oleracea (Lepidoptera) by the ectoparasitoid, Eulophus pennicornis, is associated with a reduction in host haemolymph phenoloxidase activity

When haemolymph from fifth instar Lacanobia oleracea was incubated in vitro, rapid melanization occurred. Similar levels of melanization occurred in haemolymph from larvae that had been experimentally injected with venom from the ectoparasitic wasp, Eulophus pennicornis. In contrast, haemolymph from larvae parasitized by this wasp melanized more slowly and less extensively. Phenoloxidase assays indicated that enzyme activity was present in haemocyte lysate supernatants, serum and plasma from L. oleracea and that on day 5 post-parasitization, fractions prepared from parasitized larvae had significantly less phenoloxidase activity than similar fractions from untreated or experimentally envenomated larvae. In addition, no PO activity was detectable in wasp venom, and the venom had no effect on L. oleracea plasma phenoloxidase activity in vitro. These results indicate that parasitism of L. oleracea by E. pennicornis suppresses host haemolymph phenoloxidase activity and that this suppression is not induced by adult wasp venom. The results are discussed with reference to the survival advantages of suppressing the activity of this host enzyme, and to the possible source(s) of putative suppressive factors.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

Cry1Ac杀虫蛋白对粘虫中肠几种酶活性的影响

为阐明Bt杀虫蛋白对次要靶标害虫粘虫Mythimna separata (Walker) （鳞翅目： 夜蛾科）的生理学影响, 本研究分析比较了粘虫高龄幼虫在室内取食低剂量Cry1Ac杀虫蛋白6, 12, 24和36 h后, 其体内主要的解毒酶（酯酶和谷胱甘肽-S-转移酶）、 保护酶（超氧化物歧化酶、 过氧化氢酶和过氧化物酶）和中肠蛋白酶（总蛋白酶、 强碱性类胰蛋白酶、 弱碱性类胰蛋白酶和类胰凝乳蛋白酶）等活性的变化。结果表明, 取食Cry1Ac杀虫蛋白后, 粘虫幼虫体内相关酶活力呈现不同的变化趋势： （1）酯酶、 谷胱甘肽-S-转移酶、 过氧化物酶(POD)、 类胰蛋白酶和类胰凝乳蛋白酶活力较对照显著降低（P<0.05）; （2）超氧化物歧化酶(SOD) 活力较对照显著升高（P<0.05）; （3）过氧化氢酶(CAT) 活力于6, 12和24 h显著低于对照（P<0.05）, 36 h时显著高于对照（P<0.05）。结果提示Cry1Ac杀虫蛋白主要通过抑制粘虫幼虫中肠解毒酶和蛋白酶的活性, 扰乱SOD, CAT 和POD 3种保护酶的动态平衡而干扰幼虫的正常生理代谢, 从而起到毒杀粘虫的作用。