圆斑蝰蛇毒磷脂酶A_2蛋白活性表现和分子进化探讨及菜花烙铁头、烙铁头蛇毒中Jerdonase、TmF的研究

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	圆斑蝰蛇毒磷脂酶A_2蛋白活性表现和分子进化探讨及菜花烙铁头、烙铁头蛇毒中Jerdonase、TmF的研究
其他题名	How is the enzymatic behaviors and molecular evolution of phospholipase A_2 from Daboia russellii siamensis venom? And the studies to Jerdonase, TmF from Trimeresurus jerdonii and Trimeresurus mucrosquamatus venoms
	贾永红
学位类型	博士
导师	熊郁良
	2004-07
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	蛇毒圆斑蛙蛇磷脂酶a2 异二聚体结构模拟系统进化菜花烙铁头纤溶酶烙铁头舒缓激肤增强肚
摘要	磷脂酶AZ（PLA2）是蛇毒中含量较为丰富的一类作用于梭酷键的酶。迄今为止，己有多种形式的PLA2从不同地域、不同种属的蛇毒中得以纯化并进行了较为系统的研究。其中，以VipoXin为代表的异二聚体形式PLA2较为引人注目，原因在于这种形式不同于此类蛋白家族中的诸多其它个体。目前，己经有许多关于此异二聚体PL凡生物学特性的报道，包括对此类形式存在原因、活性变化、结构表现、系统进化等方面的讨论。然而至今，这种以异二聚体形式存在的PLA2仅发现于几种蛙亚科（ViperinaeSubfamily）蛇种的蛇毒中，其中就包括我国台湾岛的圆斑蜂蛇台湾亚种（Doboiarusselliiformosensis），而蝮亚科（CrotaiinaeSubfamil）蛇种的蛇毒至今却没有此类报道。我国大陆西南端接壤东南亚，存在于云南、福建一带的圆斑蛙蛇隶属圆斑蛙蛇泰国亚种（Daboiarusselliisiamensis），那么这种蛇毒中是否也含有异二聚体形式的PLA2呢？本工作就此疑问对云南产圆斑蛙蛇泰国亚种（D.r.siamensis）蛇毒中的PLA2进行了研究，结果得到三个新的PLAZ，分别命名为DRS-PLA2-I、DRS-PLA2-II和DRS-PLA2-III。其中，DRS-PLA2-I的分子量为13864.06Da，理论pI为4.56，PLA2活性为12.35μmol/mg/min；DRS-PLA2-II的分子量为13635.99Da，理论pI为8.74，PLA2活性为8.76μmol／mg/min；DRS-PLA2-III的分子量为13619.80Da，理论厂为4.61，无PLA2活性。这三个蛋白酶N端的30个氨基酸残基恰好和三个阳性克隆的cDNA序列推导的蛋白序列吻合，结合已经报道的PLA2蛋白家族蛋白序列的保守性表现，我们可以断定它们之间存在对应关系。分子系统学分析表明DRS-PLA2-II和DRS-PLA2-III在进化关系上和蛙亚科的异二聚体PLA2关系较近，并且二者酶活性分别与异二聚体PLA2的Normalchain和Inhibitorchain相一致，只是没有发现类似Vipoxin形式的异二聚体结合蛋白。这些分析表明DRS-PLA2-nORS-PLA2-III类似圆斑蛙蛇台湾亚种（D.r.forlnos翻s沽）中的PV-4/RV-7，是PLA2异二聚体的一种特殊形式，在进化上滞后于VinOXin。另夕卜本工作还相继从云南产菜花烙铁头（Trimeresrusjerdonii）蛇毒和湖南产烙铁头（Trimeresurusmucrosquamatus）蛇毒中分离得到Jerdonase和TmF。前者为一个丝氨酸蛋白酶性质的、具有纤维蛋白原水解作用和激肤释放酶原水解作用双重活性表现的、高分子量的份五brinogenase，其活性表现可以被PMSF彻底抑制，而EDTA对此却没有影响。其它的几种抑制剂如大豆胰蛋白酶抑制剂、l-cysteine、DTT对Jerdonase的活性表现也有不同程度的影响。在Jerdonase的这些生化特性上中，分子量的大小和对纤维蛋白酶水解的特性这两方面有别于蛇毒中诸多其它来源的同类蛋白；后者T淤为一个舒缓激肚增强肤（BradykninPQtentiatingPePtide，BPP），电离质谱分析表明其分子量为1110.7Da。此小肚氨基酸序列为促进舒缓激肚（Bradki垃n，BK）诱导的豚鼠回肠纵行肌收缩的活力单位为（1.13±0.3）（m留L），T妊抑制血管紧张素转化酶（ACE）对BK水解的半数抑制剂量IC50为2μg。比较已报道的从Agkistrodon属和Bothrops属中纯化得到的BPP氨基酸序列发现：BPP的N端都是特征性的pGlu，C端为IIe-Pro-Pro，有高度的保守性。另外，TmF是Trimeresurus属中此类小肤的首次纯化。总之，本研究对国产的几种常见蛇毒中的几种常见蛋白多肤进行了一定程度的探讨和分析，和相同类别的其它蛋白、多肤比较可以看到，有许多相同的地方，也有许多不同的表现，研究结果为相应领域的深入研究提供资料和思路。
其他摘要	Phospholipase A2 (PLA2, EC3.1.1.4), which catalyzes the hydrolysis of 3-OT-phospho-glycerides at the 2-position, is abundant in snake venoms. Up to now, many enzymes have been isolated and well studied from snake venoms of different regions and various genera. Among them, the heterodimers representative by Vipoxin are much more attractive because this type shows different characterizations to other PLA2s. At present, a lot of researches have been done to them including the original reason, various activities, structure exhibition, molecular evolution and phylogenetics. However, It is interesting to find that this type of heterodimer have only been found from no venoms of the genus Crotalus but the genus Viper a including Dahoia russellii formosensis, a subspecies of the genus Daboia named Taiwan Subspecies inhabitting only in Taiwan Island. So, is there also the existence of this type of heterodimer in Daboia russelli siamensis, a subspecies of the genus Daboia named Thailand Subspecies distributing in Yunnan and Fujian provinces of Mainland China? Leading by this question, we studied the venom of D. r. siamensis captured in Yunnan province and isolated three novel PLA2 termed DRS-PLA2-I, DRS-PLA2-II and DRS-PLA2-III, respectively. The molecular weight of DRS-PLA2-I was 13864.06 Da, its theoretic p/ was 4.56, its enzymatic activity was 12.35 fimol/mg/min; the molecular weight of DRS-PLA2-II was 13635.99 Da, its theoretic p/ was 8.74, its enzymatic activity was 8.76 jumol/mg/min; the molecular weight of DRS-PLA2-III was 13619.80 Da, its theoretic p/was 4.61, it showed no enzymatic activity. Thirty amino acid residues at the N-terminus of three PLA2s determined were the same as that of protein sequences deduced by three positive clones, respectively, there existed the corresponding relationship between the N-terminal protein sequences and cDNA deduced sequences according the conservative protein sequence reported before. The phylogenetic analysis showed that DRS-PLA2-II and DRS~PLA2-III were closer to the reported heterodimers of Viperinae subfamily on a molecular evolution level, the enzymatic activity exhibition of DRS-PLA2-II and DRS-PLA2-III were also identical to the normal chain and inhibitor chain of heterdimers, respectively, except for no founding of the same type of heterdimer. All these results showed that the relationship of DRS-PLA2-II/ DRS-PLA2-III was resemble to that of RV-4/RV-7 of D. r. formosensis, which was a special type lagged behind Vipoxin on a molecular level. In addition, Jerdonase and TmF were also isolated from Trimeresurus jerdonii and Trimeresurus mucrosquamatus venoms, respectively; the former was an a-fibrinogenase with high molecular weight and double enzymatic activities including the kinin-releasing and fibrin(ogen)olytic exhibitions. Its enzymatic activities can be completely inhibited by PMSE, but not EDTA. Several other inhibitors such as soybean trypsin inhibitor, /-cysteine and DTT also influenced enzymatic activity at various degrees. Among these properties of Jerdonase, both high molecular weight and the properties of the hydrolysis to fibrinogen distinguished to many other proteases of the same kind. The latter TmF was a bradykinin-potentiating peptide (BPP) with the molecular weight 1110.7 Da by ESI-MS; the sequence of TmF was pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-He-Pro-Pro, the potentiating "umit" of TmF was (1.13 ± 0.3) (mgl), The IC5o of TmF inhibiting the hydrolysis of angiotensin-converting enzyme (ACE) to bradykinin (BK) was 2 jug When compared with other BPPs isolated from the genus AgMstrodon and the genus Bothrops, we can find the conservative N-teraiinus pGlu and C-terminus Ile-Ue-Pro. Moreover, TmF was firstly purified from the genus Trimeresurus. In a word, several enzymes have been studied from three general snake venoms; many different biochemical characterizations have been found, by which expecting to give light to more deep researches.
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/6187
专题	其他
推荐引用方式 GB/T 7714	贾永红. 圆斑蝰蛇毒磷脂酶A_2蛋白活性表现和分子进化探讨及菜花烙铁头、烙铁头蛇毒中Jerdonase、TmF的研究[D]. 北京. 中国科学院研究生院,2004.

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