Immunological localization of dopamine-beta-hydroxylase on the chromaffin granule membrane

Abstract

Publisher Summary This chapter presents a study examining immunological localization of dopamine-β-hydroxylase (DBH) on the chromaffin granule membrane. The effect of antibodies to bovine DBH on the activity of the enzyme in lysed and intact bovine medullary granules was examined. Granules were lysed by homogenization in 0.005 M Tris HCl containing 0.1% Triton X-100, and aliquots of the homogenate were incubated with either normal goat serum (NGS) or anti-DBH serum (a-DBH) at 37°C. The solutions were then assayed for DBH activity. Intact granules in isotonic sucrose medium were incubated with either NGS or a-DBH and then assayed for DBH activity. Comparable preparations of intact vesicles were incubated with either NGS or a-DBH, spun at 100,000g and the supernatant assayed for DBH activity. Incubation of lysed granules with a-DBH resulted in about 80% inhibition of the enzyme. Intact granules had about 50% of the DBH activity of lysed granules. Incubation of the intact granules with a-DBH reduced the activity to 22% of the total DBH activity. This represents a 56% inhibition of the enzyme activity of the intact granules.