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J. Biol. Chem., Vol. 275, Issue 44, 34353-34358, November 3, 2000
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From the Recent biochemical studies involving
2',7'-bis-(2-carboxyethyl)-5,6-carboxylfluorescein
(BCECF)-labeled saponin-permeabilized and parasitized
erythrocytes indicated that malaria parasite cells maintain the resting
cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump
inhibitors reduces the resting pH to 6.7, suggesting proton extrusion
from the parasite cells via vacuolar H+-ATPase
(Saliba, K. J., and Kirk, K. (1999) J. Biol.
Chem. 274, 33213-33219). In the present study, we investigated
the localization of vacuolar H+-ATPase in Plasmodium
falciparum cells infecting erythrocytes. Antibodies against
vacuolar H+-ATPase subunit A and B
specifically immunostained the infecting parasite cells and recognized
a single 67- and 55-kDa polypeptide, respectively. Immunoelectron
microscopy indicated that the immunological counterpart of V-ATPase
subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being
detected at the parasitophorus vacuolar membrane of the parasite cells.
We measured the cytoplasmic pH of both infected erythrocytes and
invading malaria parasite cells by microfluorimetry using BCECF
fluorescence. It was found that a restricted area of the erythrocyte
cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B
or bafilomycin A1, specific inhibitors of vacuolar
H+-ATPase. Simultaneously, the cytoplasmic pH of the
infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate
at 0.5 mM, an inhibitor of P-type H+-ATPase,
nor ethylisopropylamiloride at 0.2 mM, an inhibitor of Na+/H+-exchanger, affected the cytoplasmic pH
of erythrocytes or infecting parasite cells. These results constitute
direct evidence that plasma membrane vacuolar H+-ATPase is
responsible for active extrusion of protons from the parasite cells.
Vacuolar H+-ATPase Localized in Plasma Membranes of
Malaria Parasite Cells, Plasmodium falciparum, Is Involved
in Regional Acidification of Parasitized Erythrocytes*
§¶,
¶
,
, and
§§
Department of Biochemistry, Faculty of
Pharmaceutical Sciences, Okayama University, Okayama 700-8530, the
** Institute of Microbial Disease, Osaka University, Suita,
Osaka 565-0871, and the 
Department of
Physiology, Kansai Medical University, Moriguchi,
Osaka 570-8506, Japan
*
This study was supported in part by Grant-in-Aid 08281105 for Scientific Research on Priority Areas from the Ministry of
Education, Science, Culture and Sports of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by the Venture Business Laboratory of Okayama
University. Present address: Dept. of Biochemistry, Faculty of
Medicine, Okayama University, Okayama 700-8558, Japan.
§§
To whom correspondence should be addressed. Tel./Fax:
81-86-251-7933; E-mail: moriyama@pheasant.pharm.okayama-u.ac.jp.
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