Bovine splenic nerve: characterization of noradrenaline-containing vesicles and other cell organelles by density gradient centrifugation

Abstract

1. Homogenates of bovine splenic nerves were subjected to differential and sucrose density gradient centrifugation. From the low-speed supernatant a high-speed sediment (mitochondria, lysosomes, microsomes and noradrenaline (NA) vesicles) was obtained. By density gradient centrifugation of this sediment it was shown that NA vesicles are slightly less dense than mitochondria, but denser than microsomes.2. In further experiments a mitochondrial and a microsomal sediment were obtained. The mitochondrial sediment was fractionated with a short centrifugation time over a density gradient ranging from 0.6 to 1.2 M sucrose. Mitochondria (fumarase and succinate-dehydrogenase) and lysosomes (acid ribonuclease and deoxyribonuclease) sedimented to the bottom of the tube. The highest concentration of NA vesicles was found in a medium position. There was only a small amount of microsomes (glucose-6-phosphatase) present.3. The microsomal sediment was centrifuged for 150 min over a density gradient ranging from 0.8 to 1.4 M sucrose. The microsomes remained on the top of the gradient. There were also some mitochondria and lysosomes present. The NA vesicles were found in highest concentration in the middle of the gradient (at about 1.2 M sucrose).4. With the use of these two density gradients, the subcellular distribution of dopamine-beta-hydroxylase, monoamine oxidase and ATPase was studied. Dopamine-beta-hydroxylase was found to be localized in the NA vesicles. Monoamine oxidase was mainly recovered in mitochondria; a small part of the enzyme appeared to be microsomal. ATPase was present in microsomal elements.