Midgut proteases contribute to the success or failure of Plasmodium infection of the mosquito. This paper examines the reciprocal effect of Plasmodium yoelii nigeriensis on midgut trypsin, chymotrypsin, aminopeptidase and carboxypeptidase in the mosquito Anopheles stephensi. The total protein ingested and the rate of protein digestion were unaffected by the parasite, but more protein was ingested at the first than the second bloodmeal. All peptidases were unaffected by the presence of the parasite during the first gonotrophic cycle, when ookinetes were penetrating the midgut. In the second gonotrophic cycle, trypsin and chymotrypsin were unaffected by growing oocysts, but aminopeptidase activity was reduced in the midguts of infected mosquitoes. Chymotrypsin activity was depressed and aminopeptidase activity elevated during the second gonotrophic cycle. Plasmodium infection has a negligible effect on bloodmeal digestion and does not limit the availability of the protein for egg production. The significance of changes in aminopeptidase activity when oocysts are present is discussed.

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.

The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.