Incorporation of 14C-amino-acids by malaria (Plasmodium lophurae) IV. In vivo utilization of host cell haemoglobin

Sherman, Irwin W.; Tanigoshi, Linda

doi:10.1016/0020-711x(70)90033-9

Cited by 42 publications

(21 citation statements)

References 8 publications

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“…Thus, the present experiments do not support the membrane toxicity mechanism. The mode of action of CQ may be related to drug concentrations within the vacuolar space, since CQ may reach millimolar levels within the food vacuole and might directly inhibit digestive enzymes essential for the degradation of ingested host cell cytosol (31)(32)(33)(34). This could explain the observed accumulation of intact hemoglobin-containing vesicles within the food vacuoles of CQ-treated P. falciparuminfected erythrocytes (35).…”

Section: Discussionmentioning

confidence: 91%

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Yayon¹,

Cabantchik²,

Ginsburg³

1985

Proc. Natl. Acad. Sci. U.S.A.

147

105

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Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the lattereither by changing the external pH or by adding the acidotropic agent NH4Cl-led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 x 10-7 M, 1.8 x 10-7 M, and 3.3 x 10-7 M, respectively. Marked resistance to CQ (IC50 = 9.8 x 10-7 M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ attained within the acidic compartment of the parasite was correlated with inhibition of parasite growth. At therapeutic drug levels, CQ accumulation caused minor changes in the food vacuole pH, whereas at higher CQ concentrations substantial alkalinization was observed. The antimalarial activity of CQ is suggested to be exerted by the interference of the high concentrations of the accumulated drug with vital functions of the food vacuole.The development of resistance of malarial parasites to the drug chloroquine (CQ), for many years the most popular and efficient antimalarial drug, has been of growing concern (1). However, the factors responsible for drug resistance have not been identified nor has the mode of CQ action been fully elucidated. The antimalarial effect of the drug has been attributed to the ability of CQ-sensitive parasites to accumulate relatively high levels of the drug (2, 3). It has been proposed that CQ accumulation results from its binding to a putative intraparasitic receptor, ferriprotoporphyrin IX (FeP), with consequent formation of a membrane lytic agent (4). Alternatively, in analogy with the lysosomotropic effects of weak bases on animal cells (5), it was postulated that CQ accumulates to high levels in the acidic food vacuoles of malaria parasites, causing their alkalinization and interfering with vital cellular processes (6).This latter hypothesis rests on the idea that the food vacuole of an intraerythrocytic malaria parasite is an acidic compartment (7,8) whose pH is maintained by metabolic input (9) and in which CQ accumulates by virtue of its weak base properties, following transmembrane proton gradients. This has recently been demonstrated by fluorescence measurements of intravacuolar pH and determination of CQ distribution as a function of established pH gradients and their dissipation by various agents (10). However, at therapeutic levels, the effect of CQ on intravacuolar pH, as determined by CQ and methylamine distribution, was minor, thus casting doubt on the validity of vacuolar alkalinization per se as a major factor in CQ-mediated inhibition of parasite development.The mode of action of CQ and the mechanism of drug resistance in human malaria have been assessed...

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Section: Discussionmentioning

confidence: 91%

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Yayon¹,

Cabantchik²,

Ginsburg³

1985

Proc. Natl. Acad. Sci. U.S.A.

147

105

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“…4 Bestatin has been shown to have anti-malarial activity against in vitro cultures (11, 39); however, bestatin toxicity is typically assayed in the presence of exogenous pools of all 20 essential amino acids. These exogenous amino acids may compensate for the inhibition of PfA-M1 and PfLAP in parasites by allowing them to overcome a block in endogenous amino acid production.…”

Section: Resultsmentioning

confidence: 99%

“…During this replication cycle, the parasite endocytoses and catabolizes large amounts (up to 75%) of host hemoglobin to constituent amino acids (2,3). This process makes available large quantities of free amino acids for parasite protein synthesis (4) and may also modulate the osmotic environment of the host cell (5) and/or create space inside the red cell for parasite growth (6). Amino acids derived from hemoglobin catabolism are also important for the uptake of isoleucine from the extracellular environment (7).…”

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confidence: 99%

Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

Dalal

Klemba

2007

Journal of Biological Chemistry

150

186

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During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.With an estimated 2 million deaths annually to its name (1), the human malaria parasite Plasmodium falciparum continues to present an enormous public health challenge. Clinical manifestations of infection appear as the parasite replicates within host erythrocytes. During this replication cycle, the parasite endocytoses and catabolizes large amounts (up to 75%) of host hemoglobin to constituent amino acids (2, 3). This process makes available large quantities of free amino acids for parasite protein synthesis (4) and may also modulate the osmotic environment of the host cell (5) and/or create space inside the red cell for parasite growth (6). Amino acids derived from hemoglobin catabolism are also important for the uptake of isoleucine from the extracellular environment (7). As hemoglobin catabolism is essential for parasite replication and is currently a major focus of anti-malarial drug development efforts, a comprehensive understanding of the biochemistry of this pathway is an urgent priority.Hemoglobin catabolism occurs in an acidic, degradative organelle termed the food vacuole (FV) 2 or digestive vacuole. In the lumen of the FV, endopeptidases of diverse catalytic mechanism and specificity (plasmepsin (PM) I, II, and IV, histo-aspartic protease, falcipain-2, -2Ј, and -3, and falcilysin) contribute to the hydrolysis of the ␣-and ␤-globin chains to oligopeptides (8, 9). These ol...

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“…Third, the composition of the amino acid pool of infected erythrocytes is similar to the amino acid composition of hemoglobin (5)(6)(7). Fourth, the infection of erythrocytes containing radiolabeled hemoglobin is followed by the appearance of labeled amino acids in parasite proteins (8)(9)(10).…”

mentioning

confidence: 93%

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.

Rosenthal

McKerrow²,

Aikawa³

et al. 1988

J. Clin. Invest.

317

231

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To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucylamido-(4-guanidino)-butane (E-64) sufficient for the needs of the parasite (1). Second, the hemoglobin content of infected erythrocytes decreases 25-75% during the life cycle of erythrocytic parasites (2,3), and the concentration of free amino acids is greater in infected erythrocytes than in uninfected erythrocytes (4). Third, the composition of the amino acid pool of infected erythrocytes is similar to the amino acid composition of hemoglobin (5-7). Fourth, the infection of erythrocytes containing radiolabeled hemoglobin is followed by the appearance of labeled amino acids in parasite proteins (8-10).Hemoglobin degradation occurs predominantly during the trophozoite stage ofthe erythrocytic life cycle ofP. falciparum. Trophozoites ingest erythrocyte cytoplasm and transport it within vesicles to a large central food vacuole (1 1, 12), where the hemoglobin-rich cytoplasm is degraded. In the food vacuole the heme moiety precipitates and is a major component of malarial pigment (13), and globin is hydrolyzed to its constituent free amino acids. The food vacuole of P. falciparum is an acidic (14, 15), membrane-bound (1 1) compartment where proteins are degraded, and therefore it appears to be analogous to the secondary lysosomes of mammalian cells, where multiple proteinases hydrolyze proteins at acid pH (16,17).The enzymatic mechanism of globin degradation within the malarial food vacuole is unknown. In previous studies, aspartic proteinases that degraded denatured hemoglobin were isolated from malaria parasites (18-20). However, denatured hemoglobin is a nonspecific substrate that can be hydrolyzed by many proteinases, and the biological role of the aspartic proteinases cannot be determined from these studies. To determine the enzymatic mechanism of globin degradation in the trophozoite food vacuole we first studied the effects of class-specific proteinase inhibitors on intact parasites. We found that two peptide inhibitors of cysteine proteinases blocked globin degradation in the food vacuole of P. falciparum trophozoites. We also identified a cysteine proteinase of trophozoites that was inhibited by the same two proteinase inhibitors and had biochemical properties that were similar to those of the lysosomal cysteine proteinase cathepsin L. Our results suggest that the cysteine proteinase we identified has a critical role in hemoglobin degradation in the food vacuole of P. falciparum trophozoites.

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Incorporation of 14C-amino-acids by malaria (Plasmodium lophurae) IV. In vivo utilization of host cell haemoglobin

Cited by 42 publications

References 8 publications

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.

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