日本血吸虫酚氧化酶的电泳及酶活性的研究

Studies on Electrophoresis and Activities of Phenoloxidase in Schistosoma japonicum

目的 观察日本血吸虫成虫酚氧化酶 ( phenol oxidase,PO)的酶谱及其活性。方法 将 42 d活成虫置含 0 .0 5 %戊巴比妥钠的 RPMI16 40培养基中 2 3℃孵育 8h后 ,分别收集雌、雄成虫 ,匀浆、超声粉碎、高速离心取上清 (含 PO) ,再进行聚丙烯酰胺凝胶电泳 ( PAGE)及酶染色后分析其酶谱 ;同时在紫外分光光度计下检测前后 2个时点的 A488值 ( A1,A2 ) ,PO活性相对值 =( A2 - A1) / ( min· m g样品蛋白 ) ,其活性值用每分钟每毫克蛋白 A488变化值表示。设立酚酶抑制剂 (丙烯基硫脲 )组 ,在该组成虫匀浆上清中预先加入丙烯基硫脲以观察其对酚氧化酶活性的抑制效果。结果 雌虫及雄虫均表现为迁移率相同的一条主带 ;但雄虫的酶带显色反应比雌虫弱。日本血吸虫雌虫及雄虫的酚氧化酶相对活性分别为 0 .16 5 m in- 1· mg- 1和 0 .0 80 5 min- 1· m g- 1 ;加入酚酶抑制剂后 ,酶活性被明显抑制。终浓度为 5 mm ol的丙烯基硫脲对成虫酚酶活性的抑制率为 85 .0 3%。当丙烯基硫脲终浓度达 2 5 m mol时 ,酚酶活性被完全抑制。结论 不仅雌性成虫含有日本血吸虫酚氧化酶 ,而且雄性成虫也含有少量的酚氧化酶且两者酶分子相同。雄性成虫含有的少量酚氧化酶的生理意义有待于进一步研究。

Objective To observe the zymogram of phenoloxidase(PO) from the adult worms of S. japonicum and to mensurate its relative activity.Methods Forty-two days living adult worms were incubated in RPMI1640 containing 0.05% sodium Phenobarbital(8 h,23℃).Then female worms and male worms were separated.Supernatant fractions were obtained by homogenating, ultrasound fragmentation and high speed centrifugation. The zymogram of PO was analyzed by means of polyacrylamide gel electrophoresis(PAGE) and its activity was detected by UV spectrophotometer. Allyl thiourea as a inhibition of PO which inhibited the activity of PO was observed too.Results The similarity of zymogram patterns from females and males were observed, including a major band and its same migrating, but a lower color response in the male worms. The relative activities of PO in female worms and in male worms were 0.165 min -1 ·mg -1 and 0.080 5 min -1 ·mg -1 respectively. The inhibition rates of 5 mmol allyl thiourea towards the activity of PO in worms were 85.03%. The activity of PO was completely inhibited in 25 mmol allyl thiourea.Conclusions Phenoloxidase of S. japonicum exists in both female worms and male worms. Their zymogram patterns were similar in the number and migrating rate of enzyme band, but the male worms shows a lower activities. The exactly physiological role of PO in male worms waits on more research. This will add new contents to reproduction and physiology of S.japonicum.