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UDPgalactose:Ceramide Galactosyltransferase of Rat Brain: A New Method of Purification and Production of Specific Antibodies

1986; Wiley; Volume: 47; Issue: 5 Linguagem: Inglês

10.1111/j.1471-4159.1986.tb00773.x

1471-4159

Nenad Nešković, G. Roussel, J.L. Nussbaum,

Proteoglycans and glycosaminoglycans research

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°, 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 UDPgalactosexeramide galactosyltransferase.