| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
1. Purification of novel phospholipase A_2 inhibitors from the blood plasma of Chinese mamushi
We have purified three distinct types of phospholipase A_2 inhibitors (PLIalpha, PLIbeta, and PLIgamma) from the blood plasma of Chinese mamushi. PLIalpha was found to be CRD-like inhibitor that we had already reported to inhibit selectively the group-II acidic PLA_2s. PLIbeta and PLIgamma were novel inhibitors purified in the present study. The former inhibited selectively the group-II basic PLA_2s, while the latter inhibited equally all of the PLA_2s including group-I PLA_2s, group-II PLA_2s, and honey bee PLA_2 (group-III).
2. cDNA cloning of Chinese mamushi PLA_2 inhibitors
We have constructed cDNA library for the liver mRNA of Chinese mamushi, cloned cDNA for PLIalpha, PLIbeta, and PLIgamma, and determined their nucleotide sequences. As a results, PLIgamma was found to be the same type of the inhibitors that had been already purified from Thailand cobra plasma and sea snake plasma, having the similar pattern of cysteine residues to those of the proteins belonging to Ly-6 superfamily. On the other hand, PLIbeta was found to have high sequence homology to leucine-rich alpha_2 glycoprotein from human sera.
3. Interaction between PLA_2 and inhibitor
One molecule of PLIbeta was composed of three identical subunits and it bound three PLA_2 molecules. From the comparisons of the specificity of PLIbeta, some amino acid residues located in or close to the interfacial binding surface of the group-II PLA_2 were suggested to be involved in the selective binding to PLIbeta. On the other hand, the residues located in or close to the Ca^<2+> binding loop of PLA_2 were suggested to be involved in the binding to PLIgamma.
4. Purification of PLA_2 inhibitor from the sera of Elaphe quadrivirgata
PLA_2 inhibitors were found to be present in the serum of nonvenomous snakes, Elaphe quadrivirgata, and were purified. The purified inhibitor was found to be the same type of PLIgamma.
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