Bovine chromaffin cells: studies on the biosynthesis of phospholipids in chromaffin granules

We have studied the biosynthesis of chromaffin granules by labelling primary cultures of bovine chromaffin cells with either [35S]methionine or various precursors for lipids. After labelling the cells were subjected to subcellular fractionation including density gradient centrifugation. After [35S]methionine significant label (mainly represented by labelled chromogranin A) was found in the soluble proteins of chromaffin granules, whereas the membranes were relatively little labelled. However incorporation into membrane bound dopamine beta-hydroxylase and cytochrome b-561 could be demonstrated. Neither of the used lipid precursors ([3H]glycerol, [3H]choline, [3H]palmitic acid or [3H]arachidonic acid) was incorporated to any significant extent into the soluble components of chromaffin granules. Thus there is no evidence that this secretory compartment contains any lipids or acylated proteins. Incorporation of lipid precursors into the membranes of chromaffin granules was apparently low. After short chases labelled lysolecithin was not present in these organelles. However with prolonged chase times labelled lysolecithin, apparently appeared in chromaffin granules irrespective of whether the cells were stimulated or not. We can conclude that the reusable membranes of chromaffin granules have a very low lipid turnover. Lysolecithin is not transferred into these organelles during biosynthesis but is formed in them during their long life span. This formation of lysolecithin is independent of stimulation of these cells and therefore unlikely to be involved in exocytosis.