Kinetics of entry of galactolipids and phospholipids into myelin.

Abstract— Brain slices from 17 day rats were incubated with ³H]galactose and ³⁵S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of ³H]cerebroside and ³H]sulphatide in myelin showed a lag of less than 15min, while appearance of ³⁵S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of ³H]cerebroside and ^3SS]sulphatide in the non-myelin fraction and of ³H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, ³⁵S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while ³H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with 2-³H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.