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Characterization of a cytosolic aminopeptidase from Encephalitozoon intestinalis

Published online by Cambridge University Press:  08 October 2002

J. J. MILLERSHIP ,
C. L. CHAPPELL ,
P. C. OKHUYSEN  and
K. F. SNOWDEN
J. J. MILLERSHIP
Affiliation:
Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843-4467, USA
C. L. CHAPPELL
Affiliation:
Division of Infectious Diseases, University of Texas-Houston Medical School and School of Public Health, Houston, Texas 77030, USA
P. C. OKHUYSEN
Affiliation:
Division of Infectious Diseases, University of Texas-Houston Medical School and School of Public Health, Houston, Texas 77030, USA
K. F. SNOWDEN
Affiliation:
Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843-4467, USA
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Abstract

Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70·8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4·8. The enzyme was both heat (>37 °C) and cold (<0 °C) labile with an optimal activity at pH 7·2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.

Keywords

ProtozoamicrosporidiaEncephalitozoon intestinalisenzymepeptidaseaminopeptidase
Type
Research Article
Information
Parasitology , Volume 124 , Issue 1 , January 2002 , pp. 1 - 7
Copyright
© 2002 Cambridge University Press

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