球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性*

以几丁质为底物,加入基本盐培养基中,诱导球孢白僵菌（Beauveriabassiana）产生分解昆虫表皮的蛋白酶。诱导物中,蝉蜕诱导的蛋白酶总活性、比活较高,经超滤、离子交换层析、亲和层析、制备性IEF电洗脱纯化了一种有凝乳弹性蛋白酶（Pr1）活性的蛋白酶BbPr1。经SDS－PAGE电泳银染后是单带,HPLC凝胶过滤显示单峰。BbPr1为单体酶,分子量为33.6kD左右,pI为7.4。底物专一性测定显示,BbPr1能水解Phe或Leu形成的酸胺键和肽键。BbPrl可被PMSF抑制,表明其活性中心有Ser残基;BbPr1还可被胰凝乳蛋白酶抑制剂TPCK和凝乳弹性蛋白酶抑制剂TEI等所抑制;胰蛋白酶抑制剂beupeptin和Epianstatin,及胃蛋白酶抑制剂Pepstain对BbPr1活性无影响。还研究了BbPr1的最适作用pH和pH稳定性。     

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.     

胰蛋白酶的天然抑制剂

一、引言在自然界存在着一类能抑制胰蛋白酶的蛋白质。它们中固有些专一地抑制胰蛋白酶;有些除胰蛋白酶外,还能抑制其他生物活性物质,如胰凝乳蛋白酶、溶血纤维蛋白酶(plasmin)等;近年还发现一些所谓多头抑制剂(multiheaded inhibitor),它们能同时,并按一定比例地抑制胰蛋白酶和胰凝乳蛋白酶。胰蛋白酶抑制剂广泛地分布在动、植物中。这类蛋白质的生理作用,还不很清楚。在胰脏和胰液中的胰蛋白酶抑制剂据推想,此抑制剂可能使那些由胰蛋白酶原转变而来的胰蛋白酶在胰脏或其导管系统中失去活性,从而对于胰脏本身具有保护作用。显然,对它们的研究,将有助于对动、植物机体内某些     

麻蝇幼虫肠道类胰蛋白酶的分离纯化及性质(英文)

麻蝇幼虫肠液经硫铵沉淀, ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析, ＳＢＢＩ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析,分离纯化出一种分子量为 １６ｋＤ的蛋白酶。底物及抑制剂的特异性表明,该酶为类胰蛋白酶。其能够强烈地降解蛋白酶非专一底物酪蛋白和 Ｈｉｄｅ ｐｏｗｄｅｒ ａｚｕｒｅ,以及类胰蛋白酶专一底物 Ｂｚ－Ｐｈｅ－Ｖａｌ－Ａｒｇ ＮＡ, Ｂｚ－Ｐｒｏ－Ｐｈｅ－Ａｒｇ ＮＡ和Ｂｚ－Ｖａｌ－Ｇｌｙ－Ａｒｇ ＮＡ．该酶又能被丝氨酸蛋白酶抑制剂ＰＭＳＦ,类胰蛋白酶抑制剂 ＳＢ－ＢＩ和Ｌｅｕｐｅｐｔｉｎ强烈地抑制。蛋白酶在酸性环境下极不稳定,在弱碱环境（ｐＨ８．５－９．５）中活性最高。     

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。     

慈菇中两种结晶的多功能蛋白酶抑制剂的研究

由于蛋白酶抑制剂在生理、生化、病理、药理上都占有很重要的地位,特别是具有多功能的蛋白酶抑制剂被广泛应用于临床,近年来愈来愈受到各方面的重视。本文报道了从慈菇中提取两种结晶的多功能蛋白酶抑制剂,它们都具有两个活力相等的活性中心,这与其他已知的同类型植物蛋白酶抑制剂不同,有它的特殊性。(1)慈菇蛋白酶抑制剂A、B,除能抑制胰蛋白酶外,还能抑制胰凝乳蛋白酶及猪颌下腺的舒缓激肽释放酶。用酰胺、酯及蛋白等不同底物分别求出抑制剂A、B对胰蛋白酶的当量抑制比值及对胰凝乳蛋白酶的半抑制比值。两者对胰蛋白酶的抑制常数在10~(-9)～10~(-10)的范围内,抑制剂B对胰蛋白酶较之A有更大的结合力,但抑制剂A对胰凝乳蛋白酶及舒缓激肽释放酶较之B却有更明显的抑制活力。(2)用葡聚糖凝胶过滤及聚丙烯酰胺凝胶电泳分别测定抑制剂A、B的分子量均在17000左右。从抑制剂A、B对胰蛋白酶的当量抑制比值求得分子量为8500。这说明每一抑制剂分子中具有两个活力相等的活性中心。(3)测定了抑制剂A、B的氨基酸组成,二者除碱性氨基酸及天冬氨酸含量略有差异外其余皆相同,各含有两对二硫键,非极性氨基酸含量较高约占60%左右。由氨基酸组成求得最小分子量约16500。(4)用二硝基氟苯,二甲基氨基萘磺酰氯及氨肽酶M测定抑制剂A、B的N末端,都证实是天冬氨酸。     

青鱼腸胃道中蛋白酶的初步研究

(1)从淡水魚青魚腸胃道中提取出魚蛋白酶,酶制剂活力以酪蛋白作底物約为結晶胰蛋白酶的1/3。酶的前身有酶原存在,激活最适pH为8。(2)酶作用的pH偏于碱,水解酪蛋白和B-精-NH_2的最适pH都为9,酶和酶原对酸都不稳定,当pH低于4吋,酶活性和酶原激活的能力迅速丧失。(3)此酶对热很稳定,反应温度55度时,反应速度仍符合Arrhenius定律。測得水解酪蛋白和B-精-NH_2的活化能分别为14.6千卡/克分子及8.0千卡/克分子,較之胰蛋白酶水解此同一底物所需的活化能为低,尤以后者更显著,約相当于1/2值。(4)对小底物的水解以B-精-NH_2活力最高,K_m=3.3×10~(-3)M(40度pH8.7),为胰蛋白酶水解此同一底物所得K_m值的二分之一,对胰凝乳蛋白酶的底物B-酪-NH_2不作用,而对Cbz-甘-苯丙及白-NH_2却有較明显的活力。(5)半胱氨酸(10~(-3)M)和PCMB(10~(-4)M)对酶活力无影响,但能被DFP和胰蛋白酶抑制剂所抑制。DFP的抑剂随其与酶保温时間的增长而增大。lO~(-4)MDFP与酶液保溫10小时后能抑制酶活力达60%。胰蛋白酶抑制剂对此酶抑制的作用与抑制胰凝乳蛋白酶相类似。(6)从青魚腸胃道中找到有类胰蛋白酶抑制剂的存在。对魚本身蛋白酶有显著的抑制作用,对胰蛋白酶亦能抑制,但抑制能力較弱。     

麻蝇幼虫肠道蛋白酶BGP的分离纯化及性质

棕尾别麻蝇幼虫肠液经ＳＤＳ－ＰＡＧＥ后,Ｘ光片显影,呈现两条蛋白酶活性带．ＩＥＦ后,两条蛋白酶活性带的等电点分别为ｐＨ７．７和６．８．麻蝇幼虫肠液经５５％～７５％硫酸铵沉淀,以及连续两次制备等电聚焦,分离纯化出等电点约为ｐＨ７．７,分子量约为３５ｋＤ的蛋白酶ＢＧＰ．该酶能分解酪蛋白和类胰蛋白酶专一底物Ｂｚ－Ｐｈｅ－Ｖａｌ－ＡｒｇＮＡ,不能分解弹性蛋白酶专一底物ｅｌａｓｔｉｎ－ＣｏｎｇｏＲｅｄ和类胰凝乳蛋白酶专一底物Ｓｕｃ（Ａｌａ）２Ｐｒｏ－ＰｈｅＮＡ．ＳＢＢＩ,Ｌｅｕｐｅｐｔｉｎ和ＰＭＳＦ能强烈抑制其活性．专一底物和抑制剂的结果表明,ＢＧＰ是一种类胰蛋白酶．其最适反应温度为５０℃,最适作用ｐＨ为８．５．不耐高温,５０℃保温３０ｍｉｎ活性急剧下降．Ｈｇ２＋,Ｚｎ２＋和Ｃｕ２＋能抑制酶活性．Ｃａ２＋,Ｍｇ２＋对酶无激活作用,ＥＤＴＡ无抑制作用．     

浙江蝮蛇(Agkistrodon halys Pallas)蛇毒激肽释放酶Ⅰ的分离纯化及其性质的研究

浙江产蝮蛇(Agkistrodon halys Pallas)蛇毒中含有激肽释放酶,不需活化即可水解激肽原,释放激肽,并具有较弱的精氨酸酯酶的活力。粗毒经DEAE纤维素(DE-22,DE-52)和Sephadex G-75分离纯化后,可得到两个激肽释放酶的组分:Ⅰ与Ⅱ,二者电泳行为与酶活力有所不同,激肽释放酶Ⅰ在聚丙烯酰胺凝胶电泳中呈一条带,而组分Ⅱ中还杂有少量组分Ⅰ。激肽释放酶Ⅰ为一糖蛋白,含糖量20.3%,约由221个氨基酸残基组成,凝胶过滤和SDS电泳测定其分子量分别为31,000和30,000。此酶具有严格的底物专一性,能作用于激肽释放肽的专一底物Z-Phe-Arg-MCA及Bz-Pro-Phe-Arg-PA,不作用于一般蛋白质底物酪蛋白,对TAME的水解速度仅是对BAEE的14%。以BAEE为底物时,其最适pH为8～9,K_m值为2.85×10~(-4)M。本文测定了不同pH和不同温度下酶的稳定性,pH低于5或大于9,温度在40℃以上,酶活力迅速下降。其精氨酸酯酶及激肽释放酶的活力均能被丝氨酸蛋白酶抑制剂PMSF和DFP所抑制,两者呈平行关系,但都不被胰蛋白酶的专一抑制剂TLCK所抑制,慈菇抑制剂与大豆(Kunitz)抑制剂对此酶有部分抑制作用。经磷酸纤维素等阳离子交换树脂层析或交联的慈菇抑制剂Sepharose-4B亲合层析也能提纯激肽释放酶Ⅰ,但提纯后精氨酸酯酶活力下降,激肽释放活力几乎全部丧失。经圆二色光谱测定表明,酶的构象已发生改变。     

非洲爪蟾孵化酶的纯化及其部分生化特性研究

本文以ＧＳＴ－ＵＶＳ．２抗体和卵黄膜为检测手段,采用凝胶过滤和离子交换等方法将非洲爪蟾（Ｘｅｎｏｐｕｓｌａｅｖｉｓ）孵化酶纯化了９０倍以上,并研究了其酶活性和生化特性。实验发现,孵化酶分子量为６０ｋＤ,有很强的蛋白酶活性和卵黄膜溶解活性;它很不稳定,在纯化时极易降解为４０ｋＤ分子,４０ｋＤ分子没有卵黄膜溶解活性,但仍有很强的蛋白酶活性。４０ｋＤ分子很可能只代表６０ｋＤ分子中的蛋白酶功能区,而丢失了两个ＣＵＢ重复区。孵化酶对ＥＤＴＡ和金属离子非常敏感、又为ｐ－ＡＰＭＳＦ等胰蛋白酶抑制剂所强烈抑制,表明它是属于胰蛋白酶类型的一种金属蛋白酶。其特异性ＭＣＡ－底物为Ｂｏｃ－Ｌｅｕ－Ｇｌｙ－Ａｒｇ－ＭＣＡ。     

The susceptibility of MAP-2 to proteolytic degradation increases when bound to tubulin

During experiments studying dietary effects on phosphorylation/dephosphorylation of MAP-2 we found that incubation of microtubules with alkaline phosphatase resulted in extensive proteolysis of MAP-2 but not of tubulin or Tau proteins. In the absence of tubulin, when microtubule-associated proteins (MAPs) were incubated with alkaline phosphatase, MAP-2 was not proteolyzed. This suggests that binding to tubulin induces a conformational change in MAP-2 which makes it more susceptible to proteolysis. The proteolysis of MAP-2 by alkaline phosphatase was prevented by inhibitors of serine proteases, suggesting that the commercial preparation of the enzyme is contaminated by a serine protease and/or that the enzyme also has a weaker proteolytic activity. In addition, selective proteolysis of MAP-2 can be obtained with the metalloprotease collagenase. Brain homogenates are shown to contain a Ca²⁺-dependent protease which selectively degrades MAP-2 bound to tubulin. These results suggest that selective proteolysis of tubulin-bound MAP-2 could play a role in the regulation of microtubule dynamics in response to extracellular signals.     

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule-associated protein 2 (MAP-2). Activation of the N -methyl- d -aspartate (NMDA) receptor dephosphorylates MAP-2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP-2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP-2. Also, the amount of MAP-2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP-2 was decreased by ∼75% 1-2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP-2 was prevented completely by injection of 2 mg/kg MK-801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l -Carnitine, which protects rats against ammonia toxicity, also prevented MAP-2 degradation. Because activation of the NMDA receptor increases [Ca²⁺]_i, we determined whether rat brain contains a Ca²⁺-dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca²⁺-dependent protease that degrades MAP-2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca²⁺]_i, which activates calpain I. This, in turn, selectively degrades MAP-2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.     

Structural homology of microtubule-associated proteins 1 and 2 demonstrated by peptide mapping and immunoreactivity

The major high molecular weight microtubule-associated polypeptides from hog brain (MAP-1 and MAP-2) were compared by one- and two-dimensional peptide mapping under varied conditions and by immunological techniques. Partial digestion of MAP-1 and MAP-2 with Staphylococcus aureus V8 protease and analysis in one dimension gave rise to very similar peptide maps independent of whether 125I-, 3H-, or 32P-labeled proteins were used. One-dimensional cleavage patterns of significant similarity were also obtained by partial digestion of MAP-1 and MAP-2 using trypsin or chymotrypsin. Furthermore, a pronounced similarity, although clear nonidentity, of MAP-1 and MAP-2 was also revealed after exhaustive digestion of 125I-labeled proteins with S. aureus V8 protease or trypsin followed by analysis of peptides in two dimensions. For immunological comparison, antisera were used that had been raised in rabbits using electrophoretically purified MAP-1 and MAP-2 components as immunogens. As determined by immunoprecipitation, the antiserum raised to MAP-1 was equally reactive with MAP-1 and MAP-2 components, whereas the antiserum to MAP-2 reacted primarily with MAP-2. Indicating the presence of common as well as unique antigenic determinants on MAP-1 and MAP-2, these results, therefore, were in agreement with the peptide mapping data. Implications of these results for biosynthetic mechanisms as well as differential distribution and functions of MAPs in cells are discussed.     

Microheterogeneity of microtubule-associated proteins, MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents

High molecular weight microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), prepared by copolymerization with tubulin, were electrophorectically separated into three and two major subcomponents, respectively, using 5% sodium dodecyl sulfate-polyacrylamide gels. By two-dimensional gel electrophoresis, all five MAP components were shown to possess a pI of around 5. Four of these proteins, MAP-1A, MAP-1C, MAP-2A, and MAP-2B, present in comparable amounts, were iodinated after electrophoretic separation and analyzed by two-dimensional peptide mapping. With both trypsin and V8 protease, almost identical patterns were obtained from MAP-2A and MAP-2B. MAP-1A and MAP-1C, too, gave similar digestion patterns, although some differences were noted. Incubation with [gamma-32P]ATP demonstrated that endogeneous protein kinase activities phosphorylated individual subcomponents at different rates. MAP-2A, the highest labeled component, was phosphorylated 2.5-fold compared to MAP-2B both in the presence and the absence of cAMP. Labeling of MAP-1 subcomponents was 4 times less than that of MAP-2A in the absence and 16 times less in the presence of cAMP. 32P-labeled MAP-2A and MAP-2B bands were indistinguishable by one-dimensional peptide mapping, as were the three MAP-1 bands. For both MAP-1 and MAP-2 subcomponents, cAMP induced phosphorylation at new molecular sites. Incubation of radiolabeled microtubule proteins with 1 mM ATP effected, upon electrophoresis, a clear shift of MAP-2A and MAP-2B bands to positions of higher apparent molecular weights, while only slightly affecting MAP-1 bands.     

Molting fluid enzymes of the tobacco hornworm,Manduca sexta: Timing of proteolytic and chitinolytic activity in relation to pre-ecdysial development

Developmental profiles for a number of molting fluid (MF) enzyme activities were established and related to the progress of pupal cuticle degradation during the four days that precede the eclosion of adult tobacco hornworms. Cuticle degrading activity, molting fluid protease 1 (MFP-1), and molting fluid protease 2 (MFP-2) all increased in activity at the time that loss of material from the old cuticle occurred. In contrast, chitinase and β-acetylglucosaminidase activities did not parallel weight loss from the old cuticle. These results are consistent with the hypothesis that proteolytic activity is a prerequisite for the action of chitinase on cuticle chitin. © 1993 Wiley-Liss, Inc.     

Identification and spatial arrangement of high molecular weight proteins (Mr 300 000-330 000) co-assembling with microtubules from a cultured cell line (rat glioma C6)

The distribution of three high molecular weight proteins, MAP-1 (Mr 330 000), MAP-2 (Mr 300 000) and plectin (Mr 300 000) in various fractions obtained in cycles of temperature-dependent polymerization/depolymerization of microtubules from rat glioma C6 cells was studied. Using gel electrophoresis and immunoautoradiography/immunoblotting all three proteins were found to codistribute only partially with tubulin because considerable parts remained in the cold-insoluble fractions. Moreover, the proteins, particularly MAPs, were proteolytically degraded during cycling. By contrast, when microtubules were polymerized with taxol after isotonic cell lysis a considerable enrichment of MAP-1 and MAP-2 was achieved; again, plectin co-distributed only partially. In this procedure too, MAPs, especially MAP-2, were found to be highly subject to proteolysis, unless free Ca2+-ions were rigorously avoided. Proteolytic fragments generated from MAP-2 were of similar size independent of whether temperature- or taxol-dependent polymerization procedures were used, suggesting the occurrence of a MAP-2-specific protease. When the spatial arrangement of the high Mr proteins on taxol-polymerized C6 cell microtubules was directly visualized using gold-immunoelectron microscopy, a periodical, apparently helical, decoration of microtubules was found for MAP-1 and MAP-2; plectin was irregularly arrayed. A predominantly helical arrangement of both MAPs was demonstrated also for microtubules reconstituted from mammalian brain.     

Production and Regulation of Cuticle-Degrading Proteases from <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in the Presence of <Emphasis Type="Italic">Rhammatocerus schistocercoides</Emphasis> Cuticle

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.     

Cloning of a novel protease required for the molting of Locusta migratoria manilensis

Molting is required for progression between larval stages in the life cycle of an insect. The essence of insect molting is the laying down of new cuticle followed by shedding of the old cuticle. Degradation and recycling of old cuticle are brought about by enzymes present in the molting fluid, which fills the space between the old and new cuticle. Here, we describe the cloning of a novel protease gene from Locusta migratoria manilensis, designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). Sequence analysis indicated that Lm-TSP belongs to the trypsin-like serine protease family. We show, by RNA interference (RNAi), that silencing of Lm-TSP leads to dramatic reductions in protease and cuticle-degrading activity of a molting fluid, which leads to molting defects from fourth-instar larvae (L4) to fifth-instar larvae (L5), and between L5 and adult stages. These observations suggest that Lm-TSP plays a critical role in L. migratoria manilensis ecdysis.     

CD-3-mediated activation of MAP-2 kinase can be modified by ligation of the CD4 receptor. Evidence for tyrosine phosphorylation during activation of this kinase

The CD4R has been shown to exert variable effects on T cell activation responses. Depending on the manner of ligation, the CD4R has been demonstrated to have positive as well as negative effects on the generation of [Ca2+]i flux by the CD3R. Coaggregation of CD3 with CD4 enhanced Ca2+ flux while their independent ligation and aggregation diminished this response. To further elucidate these paradoxical CD4 effects, we studied induction of a microtubule-associated protein 2 kinase (MAP-2K) activity during ligation of the CD3R. Lymphoid MAP-2K activation by CD3 is an evanescent event that is dependent on phosphorylation of 43-kDa MAP-2K via a pathway that involves protein kinase C. Coaggregation of CD4 and CD3 with cross-linking antibodies and avidin enhanced the CD3-mediated MAP-2K response almost twofold. In contrast, independent ligation and cross-linking of CD4 reduced the CD3-induced MAP-2K response by approximately 50%. An important requirement for this inhibitory effect was that CD4 be ligated before stimulation with anti-CD3. The negative effect of anti-CD4 mAb was specific as other mAb failed to simulate this event. The PMA-induced MAP-2K response was not inhibited by anti-CD4. Intact 32P-labeled Jurkat and normal human T cells demonstrated the appearance of a single 43-kDa tyrosine phosphoprotein during stimulation with PMA and anti-CD3. When these crude cellular extracts were extensively fractionated across DEAE- and hydrophobic columns, MAP-2K was resolved into two peaks of activity, each containing a single tyrosine phosphoprotein around 43 kDa. In addition to tyrosine-specific labeling, mitogenic stimulation of normal human T cells also induced threonine-specific labeling of MAP-2K. These results imply that activation of lymphoid MAP-2K is a dual process requiring at least two independent kinases for optimal activity. Inasmuch as CD3 activates protein kinase C and CD4 is associated with a tyrosine kinase, pp56lck, we suggest that their coaggregation may create the conditions whereby MAP-2K may be activated by dual phosphorylation. Independent aggregation of these receptors may lead to physical separation and breakdown of this interactive mechanism.     

Antiamylase-pullulanase enzyme monoclonals which specifically inhibit amylase or pullulanase activity

Monoclonal antibodies against amylase-pullulanase enzyme from Bacillus circulans F-2 have been produced to locate and characterize the catalytic sites of the enzyme. The antibodies have been examined for inhibition of both enzyme activities of amylase and pullulanase and then classified into four types: Type I which inhibited amylase activity, Type II which inhibited pullulanase activity, Type III which inhibited both enzyme activities, and Type IV which had no effect on either enzyme activity. Only two monoclonal antibodies (MAP-12 and MAP-17) as Type I and two antibodies (MAP-3 and MAP-5) as Type II were isolated. The inhibitory activities of the antibodies were characterized and compared. In Type II antibodies, the maximal demonstrated inhibition on the pullulanase activity was 88% for MAP-3 with 1 microg of antibody and 90% for MAP-5 with 2 microg of antibody, but did not inhibit the amylase activity. In Type I antibodies, in contrast, the maximal demonstrated inhibition on the amylase activity was 94% for MAP-12 and 97% for MAP-17 with 1 microg of antibody, respectively, but no inhibition of the pullulanase was noted. MAP-12 recognized sequential epitope, while MAP-17 recognized conformation-dependent epitope of amylase activity-related regions. However, both MAP-3 and MAP-5 recognized the conformation-dependent epitope of the pullulanase activity-related region. Furthermore, the antibodies of MAP-3, MAP-5, MAP-12, and MAP-17 did not compete with one another for binding to the enzyme, indicating that they have different target epitopes on the enzyme. Antibody binding of MAP-12 and MAP-17 to the enzyme was not specifically affected by any of the antiamylase compounds tested: (a) nojirimycin; and (b) 1-deoxynojirimycin. Kinetic analysis of their effects provides evidence that both antibodies of MAP-12 and MAP-17 decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.