基质辅助激光解吸/电离质谱法研究海兔卵多肽内切酶组成、结构与功能  

Composition,Structure and Function from Endopeptidease of Aplysia Egg Analyzed with Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry

作　　者：黄河清[1] 陆永进[1] 林庆梅[1] 卓慧钦[1] 黄慧英[1] 

机构地区：[1]厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室

出　　处：《分析化学》2007年第8期1105-1110,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No30470372);厦门大学科研基金(No2004xdcx207;xdkjcx200510009)资助项目

摘　　要：选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离(MALDI-TOF)质谱技术测定AEE由单类型亚基(M)组成,其m/z比值分别为12738.17、18108.79和38221.42,即分子式为[M3+]、[M2+]和[M+]。其中AEE分子量为38221.42Da(称为AEE39),是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件,鉴定AEE39酶切INS的位点,发现易酶切位点是Leu-X(氨基酸残基),其次是Glu-X,部分Phen-X、Asn-X和Ser-X结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后,指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素,起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽(acidic pep-tide,AP)中Leu-Leu的能力,属于一种广谱性多肽内切酶。Endopeptidease of Aplysia egg （AEE） was separated and purified by column chromatography usingseparation gel both DEAE-52 cellulose. The purity of AEE and its molecular weight of the subunit （ approxi mately 39.0 kDa） were determined by Sephadex G-150 and SDS-polyacrylamid gel electrophoresis （PAGE） methods. The results from MALDI-TOF mass spectrometry indicated that AEE39 consisted of single subunit type （M）, and its ratio of mass to charge （m/z） was 12738.17, 18108.79 and 38221.42, called [ M^3＋], [ M^2＋ ], and [ M^ ], respectively. Using probe of insulin （ INS）, a combined technology with both metal che- lator EDTA and MALDI-TOF mass spectrometry was employed to measure molecular weight of AEE39 and to identify an endopeptidease with metal zinc, showing molecular weight with 38221. 42Da, called AEE39. It was found that AEE39 has the capacity for lysating INS, giving enzymolysis produces with m/z 1449.51, 2085.84, 4080. 41, 4165.42. The software designed with ourselves was used to analyze the produces sequences and to identify the cleavage of INS. As a conclusion, it was found that the best sensitive cleavage for enzymolysis was Leu （ leucine ） -X （ X ： residues of amino acid）, next one is Glu （ glutamic acid） -X. In addition, Phe（phenylalanine）-X, Asn（asparagine）-X and Ser （serine）-X in INS can be lysated once in a while. Compared to molecular structure both INS and attraction, it indicated that one of main functions of AEE39 was responsible for lysating the attraction in egg, which played an important role in informational inter- course, recalling, recognizing, and mating. In addition, AEE39 had another novel function for enzymolysis Leu-Leu in acidic peptide, which was a muhifunctional ednopeptidease.

关 键 词：海兔 多肽内切酶 质谱分析 吸引素 胰岛素 

分 类 号：Q55-3[生物学—生物化学]