| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Our previous study has revealed that, in SHR,the gene encoding the delta1 isoform of phosphatidylinositol-specific phospholipase C (PLC-delta1) has two missence base replacements in the region encoding catalytic domainwhen compared with genes from other normotensive rats such as WKY.Since a number of reports concern augmented PLC activities together with increased cellular Ca^<2+> levels or the growth rate of many cell types of SHR,it is important to clarify whether PLC-delta1 is responsible for these phenomena, and whether SHR-derived PLC-delta1 differs in enzymatic activity and/or in the secondary or the tertiary structure from that of normotensive rats. Using cloned cDNAs encodnig SHR-PLC-delta1 and WKY-PLC-delta1, we expressed each molecule in E.coli as a fusion protein with glutathione S-transferase, and found that each bacteriallysate showed high PLC activity. We were not able to detect significant difference in the specific activity, substrate specificity, Ca^<2+> requirement or
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pH dependency between affinity-purified SHR-and WKY-type enzymes. Yeast cells carrying the PLC-delta1 gene of SHR displayd accelerated cell growth in comparison to cells carrying the PLC-delta1 gene of WKY.The transformed yeast cells (SHR PLC-delta1) also showed increased levels of intracellular calcium when measured by two independent methods, namely by monitoring the absorbance ratio of indo dextran incorporated into cells by UV-laser scan microscopy and employing recombinant aequorin, a Ca^<2+>-specific indicator, to measure calcium. Affinity purified PLC-delta1 proteins from the two mutated strains of yeast cells showed nearly the same enzymic activity, whereas whole extract of cells carrying the PLC-delta1 of SHR showed higher enzymic activity than yeast cells carrying the PLC-delta1 gene of WKY.The results of the study suggest that the two amino acid replacements in the x domain of PLC-delta1 of SHR is related to the increased enzymic activity of the mutant protein, possibly involving an interaction with an unidentified modulator. It is proposed that the resultant accelerated cell growth and increased [Ca^<2+>] i may be the major cause of hypertension-related phenomena, such as hyperplasia, observed in SHR. Less
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