Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae.
Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.
Millership
Jason J
JJ
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4467, USA.
Chappell
Cynthia
C
Okhuysen
Pablo C
PC
Snowden
Karen F
KF
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D009670:Encephalitozoon cuniculi / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D021863:Vittaforma / Q000201:enzymology*
D002614:Chelating Agents; D005456:Fluorescent Dyes; D010618:Phenanthrolines; D011480:Protease Inhibitors; D004492:Edetic Acid; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex; C025205:1,10-phenanthroline