Tissue fractionation and catecholamines—II

Abstract

The intracellular localization of several enzymes involved in the biosynthesis and metabolism of the catecholamines was investigated in bovine adrenal medulla. Various fractions obtained from a total homogenate by differential centrifugation were analyzed for this enzyme content, on a complete balance-sheet basis, using enzymes and the catecholamines as markers. The results provide evidence that tyrosine hydroxylase, dopa decarboxylase and phenylethanolamine N-methyltransferase which were found in the supernatant, are not contained in the catecholamine containing granules. Furthermore, it is probable that fractionation of tyrosine hydroxylase in sucrose solutions causes adsorption in the nuclear fraction, as proved by fractionation in isotonic KC1 solutions. Maximal values of relative specific activity can be obtained in a large mitochondrial fraction for dopamine-[beta]-hydroxylase, catecholamines, monoamine oxidase and some acid hydrolases. Analysis of this fraction by centrifuging in a density gradient was required for its resolution in 3 different populations of subcellular particles corresponding, from higher to lower density, to the catecholamine and dopamine-[beta] -hydroxylase containing granules, the lysosomes and the mitochondria, respectively. In the course of uptake experiments dopamine was most rapidly taken up into the granules. From these results, a schematic model in the biosynthesis of catecholamines is proposed, suggesting the existence of an important step between decarboxylation and [beta] -hydroxylation.