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Study of Cardiotoxin isoforms from Taiwan cobra: Inhibition of protein kinase C and interaction with ATP and phosphatidylserine.
|Publication Year :||2003|
|Abstract:||眼鏡蛇毒液中含有許多不同種類的蛋白，其中最主要的兩類是心臟毒蛋白(CTX)與神經毒蛋白(NTX)。CTX是由幾個高相似度的同功異構物(isoform)所組成，它們皆為鹼性(pH>10)、小分子量(6500?7000道耳吞），且具有四個雙硫鍵緊密相繫。這些蛇毒展現多樣性的生物功能，如紅血球細胞的溶血效應、肌肉細胞的去極化與收縮、防止血小板的凝固以及抑制磷脂蛋白激?(phospholipid protein kinase)與鈉鉀離子三磷酸腺??(Na＋-K＋-ATPase)的活性。為了進一步研究CTX的結構及其生化行為，我們已完成了使用陽離子交換樹脂層析(cation exchange chromatography)與高效能層析法(reverse-phase HPLC)來純化CTX。從高效能層析管柱沖提出的純化蛋白已經由質譜儀(mass spectrometry)正確地確認每一種同功異構物。|
目前研究指出，心臟毒蛋白Ⅱ(CTXⅡ)會與四種三磷酸核?(nucleotide triphosphates)（三磷酸尿?(ATP)，三磷酸尿?(UTP)，三磷酸鳥?(GTP)與三磷酸胞?(CTP))結合，且這個現象被認為是造成蛋白激?C(PKC)與鈉鉀離子三磷酸腺??(Na＋-K＋-ATPase)活性抑制的原因。在本論文中，藉由使用酪氨酸螢光光譜(tyrosine fluorescence spectroscopy)，我們測定各個CTX isoforms與ATP結合的解離係數(dissociation constants)。此外，我們亦完成CTX抑制PKC活性的實驗。然而，我們發現與ATP結合力較佳的蛇毒對PKC卻沒有較強的抑制力，這結果指出CTX與ATP結合並不是抑制PKC活性的原因。接著進行PKC的實驗，我們進一步證實CTX與磷脂類分子(phosPholipids)，如磷脂醯絲氨酸(phosphatidylserine(PS))、二脂醯甘油(diacylglycerol (DAG))及diolein結合會造成PKC的活性被抑制。這種抑制作用主要是因蛇毒與磷脂類分子的結合會間接削弱磷脂類分子與PKC結合的能力。此外，我們亦利用圓偏光二色光譜(circular dichroism)，螢光光譜及分子模型軟體(molecular modeling)來探討CTX與PS、diolein的結合情形。根據實驗結果，我們相信CTX與磷脂類分子結合必定在抑制PKC活性的功能上扮演重要角色。最後，我們也完成了CTX Ⅲ的結晶實驗並得到初步的繞射圖譜。未來我們將以γ蛇毒(toxin γ)為模型，並嘗試利用分子取代的方法來決定其結構。
Snake venoms from the elapid family such as cobra are a mixture of many different types of proteins, of which cardiotoxins (CTXs) and neurotoxins (NTXs) are the two major toxic components. CTXs consist of several highly homologous isoforms, which are highly basic (pI>10.0), small in size (6.5?7.0 kDa) and very compact with four disulfide bonds in each molecule. These toxins display a wide variety of biological activities, such as hemolysis of red blood cells, depolarization and contraction of muscular cells, prevention of platelet aggregation, and blockage of the enzymatic activities of phospholipid protein kinase and Na+-K＋-ATPase. In order to study the structures and, in turn, the biochemical behaviors of CTXs, the purification of CTXs was carried out by cation exchange chromatography coupled with reverse-phase HPLC. The purified fractions eluted from HPLC were further characterized by mass spectrometry for the unambiguous identification of each isoform.
Previously, CTX Ⅱ was proposed to bind to all four nucleotide triphosphates (ATP, UTP, GTP, and CTP) and believed to cause the inhibitory effects of enzymatic activities, such as protein kinase C and Na＋-K＋-ATPase. By using the tyrosine fluorescence spectroscopy, we were able to identify the CTX isoforms binding to ATP with different dissociation constants. In addition, the assays for the inhibition of protein kinase C (PKC) activities with CTXs were re-evaluated. Based on our findings, however, it is found that the toxins with higher affinities towards ATP do not display stronger inhibition for PKC activities, indicating that the suppressed effect on PKC activity by CTXs is not due to binding of CTXs to ATP. Using the PKC assay, we further demonstrated that the CTXs binding to phospholipids, such as phosphatidylserine (PS), diacyiglycerol (DAG) and diolein, could account for the blockade effect of PKC activities. The inhibition effect caused by CTXs is primarily due to the ability of toxins to bind to phospholipids, leading to the reduced availability of these molecules to interact with PKC. Moreover, circular dichroism (CD), fluorescence spectroscopy and molecular modeling were used to study the binding of CTX to PS or diolein. According to our results, we believed that the binding of CTXs to phospholidis plays an important role in the inhibition of PKC activities. Finally, crystallization of CTX Ⅲ isoform has been achieved and the preliminary crystal diffraction data will be presented. The structure determination will be attempted by molecular replacement utilizing the structure of toxin γ as the molecular model for future study.
|Appears in Collections:||生化科學研究所|
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