Purification and characterization of an acid proteinase from Dirofilaria immitis worms.
An acid proteinase of Dirofilaria immitis worms was purified 437-fold by gel filtration on Sephadex G-75 followed by pepstatin-Agarose gel affinity chromatography. The enzyme with a molecular weight of 42 kDa was homogeneous as judged by both affinity chromatography and SDS-polyacrylamide gel electrophoresis. Polyacrylamide disc electrophoresis at pH 8.9, however, revealed that the enzyme was composed of five multi-forms, all carrying proteinase activity. Optimum pH of the enzyme was in the range of pH 2.8 to 3.4, and its isoelectric point ranged between 5.8 and 6.4. The purified proteinase showed a potent activity against hemoglobin and myoglobin releasing acid soluble peptides, but not free amino acids. When enzymatic properties of the proteinase was compared with mammalian cathepsin D and pepsin, D. immitis proteinase activity was reduced to about 80% of the initial activity by incubating at neutral pH and 50 degrees C for 5 min, just like cathepsin D, which remained intact. Pepsin activity was completely destroyed under the same condition. An aspartic proteinase inhibitor, 1,2-epoxy-3-(p-nitrophenoxy)propane, which inhibited pepsin by 30% at 37 degrees C for 10 min, did show little effects on D. immitis proteinase and cathepsin D. Inhibitory effect of diazoacetyl-DL-norleucine methyl ester (DAN) on D. immitis proteinase was intermediate (50% after 60 min). Immunolocalization of the proteinase in the worm tissue using its monoclonal antibodies revealed that the enzyme was localized in the intestine as well as uterine wall and some small granules of microfilariae in the uterus.
Sato
K
K
College of Medical Care and Technology, Gunma University, Maebashi.
Nagai
Y
Y
Suzuki
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004183:Dirofilaria immitis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D008322:Mammals; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D011480:Protease Inhibitors; D016282:Aspartic Acid Endopeptidases