Synthesis of galactosyl ceramide and glucosyl ceramide by rat brain: Assay procedures and changes with age

Conditions were determined for assaying whole rat brain for the galactosyl- and glucosyltransferases which form cerebrosides from ceramide and a sugar nucleotide. The brain was homogenized in water, lyophilized, and suspended in benzene together with ceramide. The mixture of tissue and substrate was evaporated to dryness with nitrogen and then incubated in a medium containing EDTA, Tris buffer, dithiothreitol, nicotinamide, Mn2+, and radioactive sugar nucleotide. In the case of UDPGlc, ATP was also added. The galactosyltransferase could use Mg2+ about as well as Mg2+ and showed a marked preference for ceramides containing a hydroxy fatty acid, while the glucosyltransferase utilized ceramides of either type equally well but was only slightly stimulated by Mg2+. The pH optimum was about 7.4 for both enzymes. Other methods tested for bringing the lipoidal substrate into contact with the enzyme yielded much lower activities.The activity of the two enzymes changed with age in different fashions during the early stage of postnatal life. Galactosyltransferase activity was low in the beginning, rising 5-fold between 8 and 16 days, while glucosyltranferase activity was high in the beginning, remaining almost constant until 16 days. Both enzymes decreased in activity over the next 40 days, reaching a plateau covering at least 300 more days.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34177/1/0000466.pd