| Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum. | |
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MedLine Citation:
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PMID: 17696875 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4-5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems. |
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Authors:
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Nectarios Klonis; Olivia Tan; Katherine Jackson; Daniel Goldberg; Michael Klemba; Leann Tilley |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: The Biochemical journal Volume: 407 ISSN: 1470-8728 ISO Abbreviation: Biochem. J. Publication Date: 2007 Nov |
Date Detail:
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Created Date: 2007-10-09 Completed Date: 2007-10-31 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 2984726R Medline TA: Biochem J Country: England |
Other Details:
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Languages: eng Pagination: 343-54 Citation Subset: IM |
Affiliation:
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Department of Biochemistry, La Trobe University, Melbourne, VIC 3086, Australia. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Endocytosis / genetics, physiology* Genes, Reporter Green Fluorescent Proteins / genetics, metabolism Hemoglobins / chemistry, genetics, metabolism* Hydrogen-Ion Concentration Plasmodium falciparum / chemistry*, genetics, physiology* Recombinant Fusion Proteins / biosynthesis, genetics Transfection Vacuoles / chemistry*, genetics, physiology |
| Grant Support | |
ID/Acronym/Agency:
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AI47798/AI/NIAID NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Hemoglobins; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins |
| Comments/Corrections | |
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