Purification and characterization of a hemoglobin degrading aspartic protease from the malarial parasite Plasmodium vivax.
Aspartic proteases of human malarial parasites are thought to play key roles in essential pathways of merozoite release, invasion and host cell hemoglobin degradation during the intraerythrocytic stages of their life cycle. Therefore, we have purified and characterized Plasmodium vivax aspartic protease, to determine if this enzyme can be used as potential drug target/immunogen, and its inhibitors as potential antimalarial drug. The P. vivax aspartic protease has been purified by a combination of ion exchange and size exclusion chromatographies and HPLC. Its properties were examined in order to define a role in the hemoglobin degradation process. The purified enzyme migrated as a single band on native PAGE and SDS/PAGE with a molecular mass of 40 kDa. Gelatin zymogram analyses revealed a clear zone of proteolytic activity corresponding to the band obtained on native PAGE and SDS/PAGE. The enzyme has an optimal pH of 4.0 and exhibits its highest activity at 37 degrees C. The enzyme is inhibited by pepstatin, but not by other inhibitors including o-phenanthroline, EDTA, PMSF or E-64, supporting its designation as an aspartic protease; its IC50 value was found to be 3.0 microM. A Lineweaver Burk double reciprocal plot with pepstatin shows that the inhibition is competitive with respect to the substrate. Ca2+ and Mg2+ ions enhance the protease activity, whereas Cu2+ and Hg2+ ions were found to be inhibitory. The pivotal role of aspartic protease in initiating hemoglobin degradation in P. vivax malaria parasite is also demonstrated.
Sharma
Arun
A
Malaria Research Centre, 22 Sham Nath Marg, Delhi-110 054, India. sharmaaru20032003@yahoo.co.in
Eapen
Alex
A
Subbarao
Sarala K
SK
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology; D010966:Plasmodium vivax / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002614:Chelating Agents; D006454:Hemoglobins; D019216:Metals, Heavy; D010436:Pepstatins; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D005780:Gelatin; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin