Purification and properties of two enzymes catalyzing galactose transfer to GM2 ganglioside from rat liver Golgi.
An enzyme activity which catalyzed the transfer of galactose from UDP-galactose to GM2 ganglioside was demonstrated in rat liver homogenate and enriched 38-fold in specific activity by preparation of Golgi membranes. This activity could be solubilized from Golgi membranes by sonication and extraction with 1% Triton X-100. The solubilized activity catalyzed the formation of GM1 ganglioside and was completely dependent upon the addition of acceptor. The rate of galactose incorporation was constant for up to 5 h at 30 degrees C. This enzyme activity was further purified by gel filtration on Sepharose CL-6B and ion exchange chromatography on DEAE-Sepharose. The elution position on gel filtration corresponded to a molecular weight for the enzyme of 38,000 which was in good agreement with that obtained by sedimentation velocity studies. Ion exchange chromatography resolved GM2 ganglioside galactosyltransferase into two species. The more basic enzyme (I) comprising 28% of the recovered activity was not retarded by the column, whereas enzyme II was eluted from the resin following the application of a salt gradient. Net purification was 120- to 140-fold for each enzyme with a total recovery of 42%. Unlike the activity in the Golgi extract, the purified enzymes I and II were labile to freezing and could be stored at -20 degrees C only in the presence of 50% glycerol. Both enzymes I and II had similar molecular weights and Michaelis constants and both had a strict requirement for Mn2+. Properties which distinguish the two enzymes included pH optima (enzyme I 7.0, enzyme II 6.0) and surfactant requirements. Neither enzyme was active following removal of Triton X-100 from the preparation. Among a series of glycolipids tested for ability to serve as substrates for galactose transfer only GM2 and asialo-GM2 ganglioside served as acceptors.
D000818:Animals; D002499:Centrifugation, Density Gradient; D005678:G(M2) Ganglioside / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D005700:Galactosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008297:Male; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats
D005732:Gangliosides; D005678:G(M2) Ganglioside; D011092:Polyethylene Glycols; D017830:Octoxynol; D005700:Galactosyltransferases; D005690:Galactose