| Co-Investigator(Kenkyū-buntansha) |
SONE Michio Gunma University School of Medicine, Assistant, 医学部, 助手 (00302464)
SUGIMOTO Hiroyuki Gunma University School of Medicine, Instructor, 医学部, 講師 (00235897)
TATEI Kazuaki Gunma University School of Medicine, Instructor, 医学部, 講師 (00192633)
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| Research Abstract |
cDNAs for three phospholipase D isozymes, rPLD1a, its splice variant rPLD1b, and a new isozyme rPLD2 were cloned from rat tissues. rPLD1a and b required Arf, Rho, and PIPィイD22ィエD2, but rPLD2 only PIP2. Endogenous phospholipase inhibitors effective at low concentrations (10-5 M) were discovered in pig colon mucosa, and were purified and identified as lysoPS, PI, and lysoPI. The levels of phospholipase D activity were increased significantly in tissues from human breast cancer and rat experimentally induced colon cancer as compared with the adjacent histologically normal tissues. Thus, phospholipase D could be used as a tumor marker and also as the target of anti-tumor drug therapy.
Phospholipase D cDNA was obtained from the embryo of Drosophila melanogaster. The predicted amino acid sequence comprised 1278 amino acid residues with a molecular mass of 145 kd with the PX and PH domains and two conserved HKD motifs. The enzyme expressed in COS cells was strongly dependent on PIPィイD22ィエD2, a
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nd was stimulated about two-fold by the addition of Arf. The gene comprising 10 exons was mapped to the right arm of chromosome II (42A16-18). In situ hybridization revealed that mRNA was expressed in egg, early blastoderm, but disappeared for some period of time in the late stage of blastoderm, and then reappeared in the imaginal stage, suggesting a role of phospholipase D in the control of early embryogenesis of Drosophila melanogaster.
Lysophospholipase van be classified into low and high molecular-weight isoforms. A novel low molecular weight enzyme was cloned and named lysophospholipase II, which also contained the GXSXG motif and belonged to the esterase family as Lysophospholipase I. In collaboration with Michel, we showed that lysophospholipase I mediates the depalmitoylation of NO synthase, suggesting a role of the enzyme in the regulation of NO synthesis. cDNA for the high molecular-weight lysophospholipase was cloned from rat liver. The enzyme significantly resembled asparaginase and contained leucine zipper and ankyrin repeat. The sequence was dissimilar to that of the low molecular-weight lysophospholipase. The transacylation mediated by the high molecular-weight enzyme was examined. It transferred the fatty acid attached to the sn-1 and -2 positions of lysophospholipids to a vacant position in the different molecule of lysophospholipid. In particular, arachidonic acid was transferred from the sn-2 position to the sn-1 position at a considerable rate to yield 1-arachidonoyl phosphatidylcholine that is a well-known precursor of anandamide. Less
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