Mueller T J, Fried B
Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA.
J Parasitol. 1999 Apr;85(2):174-80.
Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.
采用明胶底物十二烷基硫酸钠聚丙烯酰胺凝胶电泳法分析卡氏棘口吸虫和三睾棘口吸虫14日龄成虫中的蛋白酶。在pH 8.0条件下，卡氏棘口吸虫成虫显示出36 kDa和58 kDa的2条蛋白酶带，而三睾棘口吸虫成虫显示出39、64、77、96、120和168 kDa的6条带。每个物种在其排泄/分泌（E/S）产物中也显示出独特的蛋白酶条带模式。卡氏棘口吸虫的E/S蛋白酶位于36和58 kDa，而三睾棘口吸虫的E/S蛋白酶位于120和168 kDa。对卡氏棘口吸虫成虫蛋白酶的进一步表征显示，有2条带（58和66 kDa）在pH 3.0 - 4.5时具有最佳活性，3条带（38、61和96 kDa）在pH 7.0 - 8.0时活性最高。当卡氏棘口吸虫虫体提取物在pH 8.0条件下于氯化钙存在下孵育时，出现了4条低分子量带（19、21、25和30 kDa），但被乙二胺四乙酸和1,10 - 菲啰啉抑制。全虫样品和E/S产物中58和38 kDa的卡氏棘口吸虫蛋白酶带以及全虫样品中36 kDa的带被苯甲基磺酰氟抑制。通过除了最近关于核苷酸差异的研究之外还展示了蛋白酶差异，本研究有助于区分这两种具有37个领棘的相关异域棘口吸虫物种。
