Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosie,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14. 16.2] and phenylethanolamine N-methyltransferase (PMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1. 1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffln cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (S jM), (ii) a "digitonin-sensitive" cytoplasnic protein pool, which required higher concentrations of digitonin for release (10 ,uM) and included TyrOHase and PMTase, and (iii) the chromaffln granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 IAM than at 40 jaM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase eftlux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromffin granule.In epinephrine-forming cells of the adrenal medulla, the catecholamine biosynthetic pathway is composed of four enzymes, tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], aromatic amino acid decarboxylase (aromatic L-amino acid carboxy-lyase, EC 4.1.1.28), dopamine ,B-hydroxylase [dopamine l3-monooxygenase; 3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (P-hydroxylating), EC 1.14.17.1] and phenylethanolamine N-methyltransferase (PMTase; Sadenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28). Dopamine o-hydroxylase is localized in the interior of the chromaffin granule and on the internal face of the granule membrane (1), while TyrOHase, aromatic amino acid decarboxylase, and PMTase are generally considered to be "soluble" cytoplasmic enzymes and ate synthesized on free ribosomes (2-4). However, morphological and subcellular fractionation studies suggest the possible particulate localization of PMTase and TyrOHase, perhaps on the surface of the chromaffmi granule. By immunocytochemistry, both PMTase and TyrOHase have been seen to be associated with chromaffin granules (5-9), and one report shows TyrOHase to be localized on microtubules (8). In purification procedur...