Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms.
We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.
D000818:Animals; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D015801:Helminth Proteins; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors; D012550:Schistosoma mansoni / Q000201:enzymology*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D015801:Helminth Proteins; D006454:Hemoglobins; D007976:Leupeptins; D010455:Peptides; D011480:Protease Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C032854:leupeptin; D004229:Dithiothreitol