Enzymatic Synthesis of Galactocerebroside by a Galactosyltransferase from Embryonic Chicken Brain

Abstract

Abstract A galactosyltransferase that catalyzes the synthesis of galactocerebrosides (galactosylceramide) containing α-hydroxy fatty acids linked to the amino group of the sphingosine moiety has been isolated from 19- to 20-day-old embryonic chicken brain. The enzyme catalyzes the following reaction: UDP-galactose-14C + ceramide → 14C-galactocerebroside (+ UDP) Several ceramides (N-acylsphingosines) were tested as galactose acceptors, including those containing C16 and C18 saturated and unsaturated fatty acids, but the only active ceramide preparation was one containing a mixture of the α-hydroxy fatty acids 2-hydroxy C18:0 (97%) and 2-hydroxy C18:1 (2%). The reaction was stimulated by Mg++, Mn++, and Ca++. UDP-Galactose-14C could not be replaced by 14C-labeled galactose, galactose-1-P, or ADP-galactose; the Km values were 1.1 x 10-4 and 0.4 x 10-4 m for ceramide and UDP-galactose, respectively. The galactosyltransferase that catalyzes the synthesis of cerebroside is different from two other galactosyltransferases previously obtained from embryonic chicken brain, which are concerned with two steps in the synthesis of gangliosides. The UDP-galactose:ceramide galactosyltransferase activity was low in homogenates of brains from young chick embryos; in the period between 15 and 20 days of embryonic development, there was a rapid increase in activity (per mg of protein). Maximal activity was detected in homogenates from 19- to 20-day-old embryos, and was 10- to 20-fold greater than the activity in young embryonic brain. The results are in accord with the suggestion that α-hydroxy fatty acid-containing cerebrosides are synthesized via the following pathway: sphingosine → ceramide → cerebroside.