PURIFICATION AND PARTIAL CHARACTERIZATION OF CYSTEINE PROTEINASE FROMSPIROMETRA-MANSONI PLEROCERCOIDS

Spirometra mansoni plerocercoids were dissected from the tissues of na turally infected snakes (Natrix trigrialateralia). Fresh plerocercoids were incubated in medium, and excretory-secretory products (E-S) were collected. In addition, soluble proteins from lyophilized plerocercoi ds (10 mg/ml) were extracted in 0.1 M sodium acetate. Proteinase activ ity was assayed with a synthetic fluorescent substrate, l-lanyl-arginy l-7-amino-4-trifluoromethylcoumarin. Proteinase was isolated from pler ocercoid extract or E-S by diethylaminoethyl trisacryl M ion-exchange chromatography and thiolpropyl-Sepharose affinity chromatography. Thes e separations resulted in a 12.2- (extract) and 15.6-fold (E-S) purifi cation of proteinase. Sodium dodecyl sulfate-polyacrylamide gel electr ophoresis of the purified materials revealed a 28-kDa band, consistent with the apparent native molecular weight (gel filtration chromatogra phy) of approximately 35 kDa. Proteinases purified from whole extracts and E-S were compared for various biochemical characteristics; inhibi tor profiles indicated that activities from both sources are cysteine proteinases, they exhibited identical pH curves with optima at pH 5.5 and a 50% activity range at pH 4.7-8.0, they cleaved collagen chains t o 3 identical products, and they showed only minor activity toward hem oglobin. Further, the proteinase purified from plerocercoids was utili zed in immunoblots with sera from sparganosis patients. Antibody (IgG) from the infected patients, but not uninfected controls, recognized t he cysteine proteinase, suggesting that this antigen may be useful in the serodiagnosis of Spirometra mansoni infection.