The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex.
The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex.
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天蛾、天蛾的多催化蛋白酶（蛋白酶体）：催化特性以及与龙虾酶复合物的免疫学比较。
DOI：
10.1006/abbi.1995.1198
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Mykles,DL
中科院分区：
文献类型：
--
作者：
Haire,MF;Clark,JJ;Jones,ME;Hendil,KB;Schwartz,LM;Mykles,DL
蛋白酶体在真核细胞中泛素依赖性和非依赖性蛋白水解中发挥着核心作用。天蛾蛋白酶体是从幼虫体壁中纯化出来的，并对其底物特异性、对蛋白酶抑制剂的敏感性以及与针对人胎盘蛋白酶体的单克隆抗体 (mAb) 的交叉反应性进行了表征。亮肽素选择性抑制胰蛋白酶样活性 (T-L)，N-乙基马来酰亚胺抑制 T-L 和胰凝乳蛋白酶样活性，而 0.02% 十二烷基硫酸钠则分别刺激肽基谷氨酰肽水解酶、支链氨基酸优先和酪蛋白分解活性 20 倍、18 倍和 3.8 倍。所有四种肽酶活性均被 3,4-二氯异香豆素抑制。一维免疫印迹分析显示，不同组织中蛋白酶体的水平和亚基组成存在差异，节间肌和卵巢中蛋白酶体的相对水平较高，马氏小管、雄性副腺和腹神经索中蛋白酶体的相对水平较低，飞行肌和脂肪体中蛋白酶体的相对水平最低，各组织中41 kDa双联体的相对量存在差异； 22-kDa 亚基仅存在于雄性副腺中，二维聚丙烯酰胺凝胶电泳显示天蛾蛋白酶体至少含有 26 个亚基，而龙虾中含有 28 个亚基，使用四种亚基特异性单克隆抗体进行免疫学分析，鉴定出天蛾和龙虾酶中人类 zeta、C2、C3 和 C8 α 型亚基的推定同源物，四种单克隆抗体中的两种与三个或更多个天蛾亚基反应，三种单克隆抗体与两个或更多个龙虾亚基反应。此外，另外两种识别多个 α 型亚基共享表位的 mAb 表明至少 15 个（龙虾）或 16 个（天蛾）亚基是 α 型。这些结果表明，节肢动物蛋白酶体的大部分亚基复杂性是广泛翻译后修饰的结果。
The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells, The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues, The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body, The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland, Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster, Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 α-type subunits in the hawkmoth and lobster enzymes, Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of α-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were α-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.