|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1995 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1994 : ¥1,300,000 (Direct Cost : ¥1,300,000)
The unique lysophosphatidic acid PHYLPA was recently isolated from myxoamoebae of a true slime mold, Physarum polycephalum, as a specific inhibitor of DNA polymerase alpha. This lipid, composed of cyclic phosphate and cyclopropane-containing fatty acid, is the only phospholipid that inhibits DNA polymerase so far investigated. Inhibition of the enzyme is largely the result of competition between the lipid and the template DNA,but not due to a decrease in the affinity of dNTPs to the enzyme. PHYLPA inhibits proliferation of human fibroblast cells in a reversible manner. It elicits an increase in 3', 5'-cyclic adenosine monophosphate in fibroblast cells, and it also elicits inositol phosphate generation, as well as a transient rise in cytoplasmic free Ca^<2+> ion. Compared with the current knowledge about common lysophosphatidic acid (LPA), PHYLPA shows opposite effects in its mode of action on both cell proliferation of fibroblasts and DNA synthesis.
PHYLPA showed selective inhibition of
a family of DNA polymerase alpha, including DNA polymerase alpha, delta, and epsilon ; but no inhibition of DNA polymerase beta or gamma was observed. To reveal the molecular mechanism of inhibition of DNA polymerases by PHYLPA,four stereo-isomers and some other derivatives were synthesized and their effects on DNA polymerases were studied. Among 8 derivatives, PHYLPA-1 (the natural PHYLPA ; sodium 1-0- [(9'S,10'R) -9', 10' -methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-2 (sodium 3-0-[(9'S,10'R) -9', 10' -methanohexadecanoyl] -sn-glycerol 1,2-cyclic phosphate) were strong and specific inhibitors of a family of DNA polymerase alpha. But their stereo-isomers were weak inhibitors, showing an importance of stereochemistry of a cyclopropane-containing fatty acid for the inhibition. Some derivatives having no cyclopropane-containing fatty acids, palmitoyl-, oleoyl-, and palmitoleoyl PHYLPA,showed inhibition to some extent ; but 1-palmitoyl and 1-oleoyl LPA,which has no cyclic-phosphate, did not show an inhibitor activity on DNA polymerases. Hence, the inhibitory activity depends on the structure of cyclic phosphate.
In Xenopus laevis oocytes, both LPA and PHYLPA activate oscillatory Cl^- currents. PHYLPA elicited oscillatory currents only when applied extracellularly and, similar to LPA,evoked homologous desensitization. PHYLPA applied to oocytes previously desensitized by LPA failed to elicit a current, indicating that LPA completely desensitized the PHYLPA receptors. In contrast, when oocytes were desensitized by PHYLPA,LPA still evoked large currents. The lack of heterologous desensitization between PHYLPA and LPA indicates that the former acts on a distinct receptor subpopulation (s), which is also activated by LPA. Less