| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
1.Substrate specificity in the salt-activation of thermolysin : There is 10,000 times or more difference in the activity between good and poor substrates, although the degree of activation (DOA) by salt was not dependent on the substrates. The enzyme activity increases with increase in hydrophobicity of the amino acid at the cleavable bond. The difference in the hydrophobicity has no effects on the salt-activation.
2.Effects of ions : The orders of the effectiveness of cations and anions were Na^+>K^+>Li^+ and Cl^->Br^-, respectively. No relationship was observed between the size of ions and DOA,and specific ion-enzyme interaction was suggested.
3.The solubility and molecular weight of thermolysin : The solubility is 1 mg/ml in the ordinary buffers, and increased greatly by addition of salts. It is suggested that thermolysin is highly hydrophobic. The enzyme exists as a monomer.
4.Electrostatic interaction : The pH-dependence of DOA was bell-shaped. DOA was 16 at pH 7.0, and 2 at pH 6 and 9. When temperature was raised from 5 to 35ﾟC,DOA decreased from 20 to 4. DOA decreased with the addition of alcohols. The activation is controlled by the electrostatic interaction between thermolysin and ions of salts.
5.Chemical modification of tyrosines : Thermolysin contains 28 tyrosyl residues. Their ionization were changed by nitration of the tyrosyl residues and amination ofthe nitrated tyrosines. DOA was lowered by introducing negative charges onto the enzyme surface by the nitration, and was recovered to the level observed for native enzyme after amination of the nitrated tyrosines. The salt-activation is cancelled by the increase in negative charges on the enzyme surface.
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