1995 Fiscal Year Final Research Report Summary
Purification and cDNA cloning of lactosylceramide synthetase.
| Project/Area Number |
06680597
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | The Tokyo Metropolitan Institute of Medical Science |
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Principal Investigator |
HASHIMOTO Yasuhiro The Tokyo Metropolitan Institute of Medical Science, Department of Membrane Biochemistry, Chief Investigator, 生態膜研究部門, 研究員 (80164797)
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| Project Period (FY) |
1994 – 1995
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| Keywords | lactosylceramide synthetase / mouse liver / Golgi enzyme |
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| Research Abstract |
A lactosylceramide synthetase (UDP-galactose : glucosylceramide beta-1,4-galactosyltransferase) has been partially purified from mouse liver. The purification procedure involved differential centrifugation for preparation of microsomal fraction, extraction of the enzyme with Triton X-100, and sequential chromatography on UDP-hexanolamine-Sepharose, UDP-galacturonic acidhexanolamine-Sepharose, and lysoglucosylceramide-formylcellulofine. At the step of lysoglucosylceramide-formylcellulofine, the enzyme became labile. Although the enzyme was partially stabilized with glycerol and sucrose, it was hardly purified further. Various other chemicals are now tested for stabilizing the enzyme. The enzyme was partially purified several thousands-fold with a specific activity of 11 nmol/mg/protein. The Km values for glucosylceramide and UDP-galactose were 7muM and 200muM,respectively. The enzyme did not bind to alpha-lactalbumin Sepharose column, indicating that the enzyme is different from lactose synthetase.
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