| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Pyrimidines are essential compounds in uncleic acid structure and function. They can be produced by the de novo and salvage biosynthetic pathways. In view of the basic importance and relative lack of understanding of initial steps of the de novo pathway in parasitic protozoa, the present investigation was undertaken to elucidate comparatively the kinetic and regulatory properties of the initial three enzymes, CPS II (carbamoyl-phoshate synthetase II), ACTase, and DHOase, in culture forms of Crithidia fasciculata. This organism belongs to the family Trypanosomatidae and we used it as a model material.
Ammonium sulfate fractionation of C. fasciculata extracts resulted in a separation of CPS II from ACTase. Recently, other laboratories also demonstrated the possible, separated nature of these enzymes in Toxoplasma gondii and Leishmania donovani. Therefore, CPS II in these unicellular eukaryotes is unique, when compared with yeast bifunctional CA protein and with multifunctional CAD protein
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in multicellular animals including helminth parasites and mammals. We first reported the details of kinetic and regulatory properties of the Crithidia CPS II that distinguished the protozoan enzyme from prokaryotic and multicellular eukaryotic enzymes. Some properties, but nt all, of the Crithidia enzyme resembles those of prokaryotic enzymes.
Acivicin, an L-glutamine antagonist with a significant antitumor activity, competitively inhibited the Crithidia CPS II activity with respect to the substraqte L-glutamine. In the absence of glutamine, however, the compound yielded a selective, time-dependent, irreversible inactivation of the glutamine-dependent CPS II activity. Thus, acivicin is an active sitedirected affinity analog of glutamine, bringing about an affinity labeling of the glutaminebinding site of the enzyme. Acivicin markedly inhibited the C. fasciculata growth in a serumfree medium GIT, and this inhibition may be primarily attributed to the possible strong inhibition (inactivation) of GMP synthetase and secondry of CTP synthetase, The mechanism of in vivo inactivation of CPS II is the same as in vitro. Less
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