Purification and partial characterization of cysteine proteinase from Spirometra mansoni plerocercoids.
Spirometra mansoni plerocercoids were dissected from the tissues of naturally infected snakes (Natrix trigrialateralia). Fresh plerocercoids were incubated in medium, and excretory-secretory products (E-S) were collected. In addition, soluble proteins from lyophilized plerocercoids (10 mg/ml) were extracted in 0.1 M sodium acetate. Proteinase activity was assayed with a synthetic fluorescent substrate, carbobenzoxy-phenylalanyl-arginyl-7-amino-4-trifluoromethylcoumarin. Proteinase was isolated from plerocercoid extract or E-S by diethylaminoethyl trisacryl M ion-exchange chromatography and thiolpropyl-Sepharose affinity chromatography. These separations resulted in a 12.2- (extract) and 15.6-fold (E-S) purification of proteinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified materials revealed a 28-kDa band, consistent with the apparent native molecular weight (gel filtration chromatography) of approximately 35 kDa. Proteinases purified from whole extracts and E-S were compared for various biochemical characteristics; inhibitor 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 to 3 identical products, and they showed only minor activity toward hemoglobin. Further, the proteinase purified from plerocercoids was utilized in immunoblots with sera from sparganosis patients. Antibody (IgG) from the infected patients, but not uninfected controls, recognized the cysteine proteinase, suggesting that this antigen may be useful in the serodiagnosis of Spirometra mansoni infection.
Song
C Y
CY
Department of Biology, Chung-Ang University, Dong Jak-ku, Seoul, Korea.
Chappell
C L
CL
D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D008970:Molecular Weight; D012911:Snakes; D013031:Sparganosis / Q000276:immunology; D013032:Sparganum / Q000201:enzymology*
D000909:Antibodies, Helminth; D006454:Hemoglobins; D007106:Immune Sera; D007074:Immunoglobulin G; D003094:Collagen; D003546:Cysteine Endopeptidases