| Research Abstract |
The most virulent form of malaria is caused by the protozoan, Plasmodiumfalciparum, which is transmitted to humans by the bite of Anopheles mosquito. Key challenges to combating this devastating disease are the spread of resistance to antimalarial drugs. We pay attention to a fact that H^+-pyrophosphatase (H^+-PPase) exists in these protozoa but not in animal cells. Thus, inhibitors of H^+-PPase are expected as a good candidate of antimalarial drug. During this study, we prepared water-soluble and organic-solvent soluble fractions from 145 marine organisms, and examined their effects on H^+-PPase. We found that a few compounds in the water-soluble fraction prepared from a soft coral inhibited H^+-PPase. These are acylspermidine derivatives, which are N',N",N"-trimethylspermidines athat are acylated by a methylbranched unsaturated fatty acid. Acylspermidines inhibited PPi hydrolysis activity and PPi-dependent H^+ translocation (half inhibition concentrations, 1 μM). These compounds actually inhibited H^+-PPase and prevent acidification of vacuoles in living protoplasts.
We also obtained the following new information on H^+-PPase.
(1) Mutant Arabidopsis plants that lack the H^+-PPase gene were extremely small. This observation indicates that H^+-PPase is essential for normal growth of plants.
(2) Two essential domains, which are involved in PPi hydrolysis, were determined by site-directed mutagenesis of mung bean H^+-PPase.
(3) Molecular properties of the PPi-dependent H^+ current through H^+-PPase were directly determined by patch clamp analysis.
(4) We established experimental conditions for expressing Streptomyces H^+-PPase in E.coli cells with a high efficiency to investigate the structure-function relationship of the enzyme.
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