UDPgalactose:Ceramide Galactosyltransferase of Rat Brain: A New Method of Purification and Production of Specific Antibodies |
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Authors: | N M Neskovic G Roussel J L Nussbaum |
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Institution: | Centre de Neurochimie du CNRS and U. 44 de l'INSERM, Strasbourg, France |
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Abstract: | A new method for purification of UDPgalactose:ceramide galactosyltransferase (EC 2.4.1.45) is described. The principal steps involved solvent extraction at -70 degrees C, Triton X-100 extraction, and DEAE-Sephadex and Blue Sepharose chromatography. The active configuration of the enzyme was stabilized by phospholipids and a rapid loss of enzymatic activity was observed after removal of these lipids. The inactive enzyme could be fully reactivated in the presence of brain phospholipids dispersed in a Triton X-100-containing buffer. The purified enzyme preparation showed two major components by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with apparent molecular weights of 50-70,000. The 53,000-dalton protein was isolated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate and used to produce antibodies against UDPgalactose:ceramide galactosyltransferase. |
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Keywords: | UDPgalactoserceramide galactosyltransferase Purification Synthetic substrate Stabilization by phospholipids Specific antibodies |
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