Dopamine beta-hydroxylase (DBH) is a catecholamine biosynthetic enzyme present inside chromaffin secretory vesicles of adrenal medulla, sympathetic nerve endings, and noradrenergic neurons of the brain. Serum also contains DBH activity. Within the secretory vesicles DBH is found in two forms, soluble and membrane-bound. The mechanism of membrane attachment and regulation of the ratio of the two forms of DBH is not understood. Analysis of purified membranous DBH has shown that it has phosphatidylserine specifically and tightly bound and that approximately one quarter of this form has uncleaved signal sequence. Both of these characteristics may influence membrane attachment. In the proposed work we will test the hypothesis that phosphatidylserine regulates the membranous to soluble ratio of dopamine beta-hydroxylase. We propose that the expression of the mature processed DBH in Drosophila melanogaster Schneider II cells will result in some of that enzyme becoming membrane-bound due to non-covalent interactions with phosphatidylerine. Further, we propose that this interaction can be directly tested by depleting the Schneider cells of phosphatidylserine. The results of the proposed work will increase our knowledge of membrane lipid-protein interactions, especially for the growing class of proteins which bind to biological membranes via interaction with phosphatidylserine. In addition, we do not understand what regulates the levels of serum DBH. The ratio of soluble to membranous forms of this enzyme may play a significant role in this parameter of clinical interest.
