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1.
尖吻蝮蛇毒磷脂酶A2基因的克隆与序列分析   总被引:6,自引:3,他引:3  
从尖吻蝮蛇毒腺中抽提总RNA,经RTPCR扩增磷脂酶A2的基因,以江浙蝮蛇的酸性磷脂酶A2基因为探针杂交筛选克隆,分离得到4种磷脂酶A2基因。经双向测序测定了这些磷脂酶A2同功酶基因的全序列,并由此推导出编码的氨基酸序列。运用计算机软件推算了它们的等电点,按照等电点和结构特征将它们分别命名为尖吻蝮蛇毒酸性磷脂酶A2I(A.aAPLA2I)、尖吻蝮蛇毒酸性磷脂酶A2II(A.aAPLA2II)、尖吻蝮蛇毒碱性磷脂酶A2(A.aBPLA2)和尖吻蝮蛇毒Lys49磷脂酶A2(A.aLys49PLA2)。其中A.aAPLA2I的1~10位氨基酸残基序列同以前分离得到的尖吻蝮蛇酸性磷脂酶A2已测定的1~10位氨基酸残基序列完全一致。A.aLys49PLA2基因则由于其推导出的49位氨基酸残基由Lys代替了Asp而区别于以前克隆到的磷脂酶A2基因。最后运用计算机软件比较了它们的同源性。这一组磷脂酶A2基因的克隆,将为进一步研究磷脂酶A2的结构与功能的关系提供更多的信息。  相似文献   

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我们用同源模建方法构建了江浙蝮蛇毒碱性-酸性杂合磷脂酶A2-Ⅱ和中性磷脂酶A2的三维结构,并对它们的结构特征做了比较分析。在此基础上,我们在三维结构水平上解释了磷脂酶A2荧光光谱学研究的一些结果。我们还对四种江浙蝮蛇毒磷脂酶A2的酶活性部位的静电势分布做了分析。我们认为:含钙的PLA2酶活性部位周围的静电势有利于PLA2与带负电荷的底物结合。  相似文献   

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江浙蝮蛇(AgkistrodonhalysPalas)是属于蝮科蝮属的有毒蛇。蝮蛇毒成份比较复杂,但其中所含的蝮蛇神经毒素(agkistrodotoxin,ATX),是主要的神经毒性成分,它具有磷脂酶A2(PLA2)活性,是一种阻碍神经肌肉接头传递的...  相似文献   

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钙离子在江浙蝮蛇毒酸性磷脂酶A_2中的作用是多方面的。它不仅能够引起酸性磷脂酶A_2在溶液中构象的变化,而且对该酶活性有较大的影响。Ca~(2+)为酶活力所必需,当Ca~(2+)浓度达到0.06mmol/L时,酶表现出很高的活力;Ca~(2+)浓度超过0.5mmol/L时,催化反应出现一个明显的延滞期。化学修饰表明His_(47)在表现活力方面起重要作用,Ca~(2+)的存在可降低其修饰反应的速度,提示这是由于Ca~(2+)引起构象发生变化而造成的。  相似文献   

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将尖吻蝮蛇毒酸性磷脂酶 A2 I( A.a A P L A2 I) 的基因克隆至表达载体p B L M V L2 , 在大肠杆菌 R R1 中成功表达。表达产物 A.a A P L A2 I约占细菌蛋白质总量的30 % , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 F P L C Superose T M12 纯化, 产物经过 S D S P A G E 检测只有单一条带。对表达的 A.a A P L A2 I进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 A.a A P L A2 I的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 A2( A P L A2) 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 A2( P L A2) 的结构与这些活性的关系进行了讨论  相似文献   

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江浙蝮蛇磷脂酶A2基因的多样性研究   总被引:4,自引:4,他引:0  
我们利用简并引物从江浙蝮蛇腺总RNA经RP-PCR扩增磷脂酶A2(简称PLA2)基因,并以碱性PLA2(B-PLA2)基因为探针,分离出了酸性PLA2(A-PLA2)和两个未见报道的特征结构类同的基因,分别命名为Asn^48-PAL2和BA-PAL2。双向测序测定了这组PLA2同工酶(除信号肽外)基因的全序列,并由此推导编码的氨基酸序列。其中A-PLA2基因编码的氨基酸序列与较早报道的由蛇毒中分离  相似文献   

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竹叶青蛇毒磷脂酶A2的分离纯化和性质研究   总被引:4,自引:0,他引:4  
竹叶青蛇毒磷脂酶A_2的分离纯化和性质研究冯波,吴卫甲,钱嵘,王克夷,周元聪(中国科学院上海生物化学研究所,200031)关键词竹叶青蛇毒,磷脂酶A_2;血小板聚集磷脂酶A。在哺乳动物胰脏和蛇毒毒液中含量较丰富,其中蛇毒磷脂酶A。除能水解甘油磷脂的第二...  相似文献   

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江浙产蝮蛇毒提取的抗凝成份激活人血浆蛋白C的作用(摘要)瞿国伟,顾肃敏,黄美华,任彤,邵慧珍1.浙江医科大学2.上海市瑞金医院本文从江浙产蝮蛇粗毒中提取到的抗凝成份(SVPA),具有激活人血浆蛋白C活性,利用被激活的蛋白C特异性地分解生色底物而生成颜...  相似文献   

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江浙蝮蛇毒酸性磷脂酶A2抑制制血小板作用的机理   总被引:7,自引:3,他引:4  
江浙蝮蛇毒酸性磷脂酶A2(APLA2)对多种致聚剂引起的血小析具有抑制作用,这种抑制并非是由于APLA2与血小板膜结合,引起膜流动性降低造成。APLA2不裂解血小板,电镜观察表明它能抑制血小析部颗粒的中心化,使之不易被激活。进一步研究显示APLA2是作用在骨架上,使致聚剂引肌动蛋白单体聚合难以进行,APLA2导致的cAMP浓度的升高正说明了这点。结果提示APLA2首先作用于轿小板细胞膜,引起cAM  相似文献   

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哺乳动物细胞液磷脂酶A2   总被引:1,自引:0,他引:1  
哺乳动物细胞液磷脂酶A_2杨在清,甘莉(华中农业大学,武汉430070)关键词细胞液磷脂酶A_2细胞液磷脂酶A2(cytosolicphospho-lipaseA2,cPLA2)是动物非胰腺磷脂酶A2的一种,存在于哺乳动物的细胞液中,在花生四烯酸的代谢...  相似文献   

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K. Hausmann 《Protoplasma》1979,100(2):199-213
Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.  相似文献   

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Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices  相似文献   

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Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418  相似文献   

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This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.  相似文献   

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