|Budget Amount *help
¥7,700,000 (Direct Cost : ¥7,700,000)
Fiscal Year 1997 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥5,400,000 (Direct Cost : ¥5,400,000)
Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. In the present study, we examined the property of PLC isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimer's disease (AD) brains. Chromatofocusing profile of human brain PLC activity on a Mono P HR column demonstrated that PLC-gamma1 exhibiting isoelectric point values of 5.2 and PLC-delta1 exhibiting isoelectric point values of 5.2 and 4.6 are partly overlapped in their elution. In contrast, the elution profile of control and AD brain PLC on a Superdex 200 pg column gel filtration chromatography indicated that PLC-gamma1 and PLC-delta1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in human brain. Using this gel filtration chromatography it was revealed that the PLC-gamma1 activity was significantly decreased and the PLC-delta1 activity was significantly increased in AD brains compared with controls. These results suggest that the PLC isozymes are differentially involved in AD.
PLC-delta1 binds to both phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P_2) and inositol 1,4,5-trisphosphate (Ins (1,4,5)P_3) with a high affinity via its pleckstrin homology (PH) domain. We found a missense mutation in the region encoding the C-terminal PH domain of the human PLC-delta1. This is also the first report of a mutation in the human PLC genes. A single base substitution (G to A) causes the amino acid replacement, Arg105 to His.Site-directed mutagenesis of the glutathione-S-transferase (GST)/PLC-delta1 fusion protein changing Arg105 to His resulted in a four-fold decrease in the affinity of specific Ins (1,4,5)P_3 binding and a reduction in PtdIns (4,5)P_2 hydrolysing activity to about 40% of that of the wild-type enzyme. This remarkable loss of function can be interpreted in terms of a conformational change in the PH domain.