Evidence that most of the dopamine β-hydroxylase is not membrane bound in purified large dense cored noradrenergic vesicles

The compartmentalization of dopamine β-hydroxylase was studied in a purified fraction of large, dense-cored noradrenergic vesicles from bovine splenic nerve. A correlative biochemical and morphological approach was used to evaluate physical methods designed to produce more efficient vesicle lysis than has been accomplished previously with freeze-thawing, hypo-osmotic shocks or incubation at 37°C, all of which are relatively ineffective. Of the seven purely physical techniques tested, only those which subjected the vesicles to high pressures followed by rapid decompression produced effective lysis, unmasking of latent enzyme activity, and release of enzyme into the soluble phase. Based on results with the French Press, the best estimate for the percentage of dopamine β-hydroxylase which can be released into the soluble phase is 55–70% of the total enzyme activity produced by the addition of 0.05% v/v Triton X-100. This includes two-thirds of the enzyme which was originally in a latent form. The enzyme is considered to occur primarily, if not entirely, in a granular complex in the vesicle matrix. About one-third is thought to be oriented specifically at the inner surface of the vesicle membrane and is active enzymatically.