| Trimeresurus albolabris snake belongs to Crotalinae Trimeresurus, which is a kind of rather poisonic snakes and it mainly distributes in Chongqing, Sichuan, Guizhou, Fujian, Guangxi, Guangdong, Hainan and Taiwan. Our laboratory did first researches on the venom of T. albolabris snakes from Jinfu mountain in Chongqing and found their venoms could inhibit platelet aggregation and then measured 5'-nucleotidase activity in the crude venom by the method of Jesudium.5'-nucleotidase is one of the phosphoesterases mainly distributed in animal cells, plant cells, bacterium and the venom of some animals. It hydrolyzed 5'-mononucleotide into nucleosides and as a tool it had been used in genetic engineering and nucleic acid research. Simultaneously, this enzyme had significant biological functions, which played vital roles in cell growth and development, movement, fibrin synthesis, neurotransmission, barrier function of epiderm or endodermis, lymphocytic adhension, blood circulation and immune response. The physicochemical properties, enzymatic properties, and biological functions of 5'-nucleotidase presented different in the different organism and different tissues in one organism, especially ones of the 5'-nucleotidase isolated from different snake venom did so. For example, the 5'-nucleotidase isolated from T. gramineus could inhibited platelet aggregation induced by ADP, AA, collagen, low concentration thrombin and Ionophor(A-23187). While 5'-nucleotidase isolated from T. Stejnegeri snake venom could inhibit platelet aggregation induced by ADP, arachidonic acid (AA), Trimeresurus mucrosquamatus venom aggregator (TMVA) and thrombin, but could disaggregated the platelet aggregation induced by ADP conspicuously. Dhananiaya et al(2006) reported that 5'-nucleotidase in the venom of Naja naja could anticoagulated human blood. Up to now, researches on 5'-nucleotidase in the venom of T. albolabris in our country did not yet reported. Therefore, we did researches on the venom of T. albolabris came from Jinfu mountain in Chongqing , isolated 5'-nucleotidase and studied its physicochemical properties, enzymatic properties and biological functions of inhibit platelet aggregations.The main results were shown below: 1. Collection and preparation of crud venoms of T. albolabris snake, which contains about 81% protein and its LD50 is 7.01±0.55 mg/kg. We detected the activities of arginine esterase was 0.536μmol /min mg, phospholipase A2 activity was 1.03μmol /min mg, 5'-nucleotidase activity was 9.02μg Pi/min mg when using AMP as substrate. Besides that alkaline phosphomonoesterase activity was 8.45μmol /min mg and phosphodiesterase activity was 17.5μmol /min mg.2. A 5'-nucleotidase was isolated and purified from the venom of T. albolabris by three steps of chromatography including DEAE-SephadexA-25, Sephadex G -100 and CM-Sephadex C-50. The purified 5'-nucleotidase was proved to be homogenous on SDS-PAGE and HPLC column chromatography. It is a glycoprotein with an apparent molecular weight of 48.03 kDa. The enzymatic activities of the purified 5'-nucleotidase were 330.33μg Pi/min mg and 123.56μg Pi/min mg when using AMP and ADP as substrates, respectively. The activities of arginine esterase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A2 were not detected. EDTA could inhibit the 5'-nucleotidase activity. Its optimum pH is 9 and its optimum temperature is 50℃. Our results indicated that 5'-nucleotidase isolated and purified from the venom of T. albolabris had many similar biochemical and enzymatical properties with other 5'-nucleotidases from different species.3. 5'-nucleotidase isolated and purified from the venom of T. albolabris could inhibit platelet aggregations induced by ADP, arachidonic acid(AA) and PAF. The inhibition effects were proportional to the concentrations of 5'-nucleotidases. The inhibitory study on washed platelets indicated that inhibitory effect of the 5'-nucleotidase on platelets aggregation was associated with removal of ADP and accumulation of adenosine.
|
PDF Full Text Request