| Project/Area Number |
12672130
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Niigata University of Pharmacy and Applied Life Science |
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Principal Investigator |
UDA Yutaka Faculty of Pharmaceutical Science, Niigata University of Pharmacy and Applied Life Sciences, Professor, 薬学部, 教授 (90013937)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAISHI Takayuki Faculty of Pharmaceutical Science, Niigata University of Pharmacy and Applied Life Sciences, Assistant Professor, 薬学部, 助教授 (40110663)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | sialidase / β-galactosidase / protective protein / β-galactosidase complex / molecular interaction / carboxypeptidase |
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| Research Abstract |
Recent studies has shown that the mammalian lysosomal sialidase exists as a complex with β-galactosidase and protective protein. The protective protein is a 48kDa heterodimer of 30kDa- and 20kDa protomers and is required for multimerization of β-galactosidase as well as the expression and stabilization of sialidase activity. To understand the function of β-galactosidase complex, we examined the molecular interaction between β-galactosidase components in the complex using Biacore surface plasmon resonance. Each of the β-galactosidase components was separated and purified as described previously. The biotinized β-galactosidase monomer(64kDa) was coupled to a streptoavidine group on the senser tip surface. The 48kDa, 30kDa and 20kDa protein components in protective protein were tested for their interaction with β-galactosidase monomer fixed on the sensor tip. Among the components, 48kDa and 20kDa proteins had strong affinity with β-galactosidase monomer at acidic pH, but 30kDa protein did not show any interaction. This result suggests that β-galactosidase bind to 20kDa protein component in protective protein and forms a β-galactosidase hetero polymer complex, in contrast with 30kDa protein which possess carboxypeptidase activity. To elucidate the molecular interaction between sialidase and β-galactosidase complex, further studies are necessary.
We highly purified a sialidase from the ovary of the starfish, Asterina pectinifera. The analysis of the N-terminal amino acid sequences of the purified enzyme showed high homology with that of cathepsin D. On the purification processes, the specific activities of not only sialidase activity but also cathepsin D activity were increased. The two enzymes showed distinct protein band on polyacrylamide gel electrophoresis. It is necessary to study more precisely whether sialidase interact with cathepsin D or not.
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