The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes.
Intra-erythrocytic Plasmodium parasites digest host cell haemoglobin and use the liberated amino acids for protein synthesis. Although several endoproteases (aspartic, cysteine, and metallo-) have been shown to be involved in the initial stages of haemoglobin degradation, little is known about the steps immediately before amino acid release. In our studies, fluorometric enzyme assays indicated that the stage of the P. falciparum erythrocytic cycle with highest aminopeptidase activity was the stage at which most haemoglobin degradation occurs, i.e. the trophozoite. Consistent with these results, metabolic growth assays indicated that the late ring/trophozoite stage was most susceptible to aminopeptidase inhibitors. To reconstitute the terminal stages of haemoglobin breakdown in vitro, we synthesised three peptides with amino acid sequences corresponding to known products of the endoproteolytic digestion of haemoglobin and employed them as substrates for aminopeptidases. Both trophozoite cytosolic extract, and partially-purified aminopeptidase, hydrolysed these peptide fragments to amino acids. Hydrolysis appeared to occur sequentially from the amino-termini of the peptides, and was inhibited in a concentration-dependent manner by the aminopeptidase-specific inhibitor nitrobestatin. The results suggest that P. falciparum aminopeptidases could be the enzymes responsible for the hydrolysis of haemoglobin-derived peptides to free amino acids. Lack of ultrastructural change in parasites treated with relevant concentrations of aminopeptidase-specific inhibitors, however, indicated that little feedback exists whereby the inhibition of cytosolic aminopeptidases results in obvious inhibition of initial haemoglobin degradation in the digestive vacuole.
Gavigan
C S
CS
Department of Microbiology, Moyne Institute, Trinity College, 2, Dublin, Ireland
Dalton
J P
JP
Bell
A
A
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D004912:Erythrocytes / Q000469:parasitology*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016778:Malaria, Falciparum / Q000469:parasitology; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity*
D006454:Hemoglobins; C114434:nitrobestatin; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex