UDPgalactose:ceramide galactosyltransferase of rat brain: a new method of purification and production of specific antibodies.
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.
D000818:Animals; D000918:Antibody Specificity; D001921:Brain / Q000201:enzymology*; D004789:Enzyme Activation; D005700:Galactosyltransferases / Q000276:immunology / Q000302:isolation & purification*; D051147:Ganglioside Galactosyltransferase; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats
D010743:Phospholipids; D005700:Galactosyltransferases; C511215:Ugt8 protein, rat; D051147:Ganglioside Galactosyltransferase