On the soluble phase of adrenergic nerve vesicles correlation of matrix density and biochemical composition

Abstract

Highly purified sympathetic nerve vesicles isolated from bovine splenic nerves were treated by hypo-osmotic shocks, freeze-thawing or incubation in the absence or presence of ATP and MgCl. The vesicle preparations were then studied morphologically by electron microscopy and their content of noradrenaline (NA), and soluble proteins analyzed biochemically with special regard to dopamine beta-hydroxylase (DBH). Hypo-osmotic shocks released about 25 per cent of the NA and protein content and about 8 per cent of the DBH activity. This treatment induced swelling of the vesicles but their membranes remained unruptured and they still contained dense cores. Freeze-thawing released about 35 per cent of the NA, 25 per cent of the proteins and 11 per cent of the DBH. After the latter treatment some matrix material still remained in most vesicles but many were less stainable than the intact vesicles in cold control preparations. During incubation at 30 degrees C in an isotonic sucrose-phosphate medium for 30 min the vesicles released most of their NA and soluble DBH activity as well as much of their matrix density. After incubation at 37 degrees C for 30 min most vesicles appeared translucent. After incubation at 30 degrees C for 30 min in the presence of ATP and MgCl the vesicles lost most of their original NA content but retained their DBH activity and most of their matrix density. The results indicate that there is not always a correlation between NA content and retention of matrix density which suggests that DBH might be a component of a macro-molecular complex responsible for the staining reaction taking place in the maxtrix of NA depleted vesicles. This hypothesis is further supported by the finding of striking similarities between DBH isolated from chromaffin granules and the granular and fibrillar material surrounding the nerve vesicles after depletion.