a b o r a t o r y of N e u r o c h e m i s t r y , N a t i o n a l I n s t i t u t e of M e n t a l H e a l t h , B e t h e s d a , Maryland the first step in the biosynthesis of norepinephrine and epinephrine involved the conversion of tyrosine to 3,4dihydroxyphenylalanine (dopa), followed by decarboxylation of the dopa to dopamine, which was then hydroxylated in the p position of the side chain and methylated on the amino group to form norepinephrine and epinephrine, respectively (Fig. 1). Although the evidence in favor of the pathway proposed by Blaschko continued to mount (5-8), and it is now widely accepted as the only quantitatively important one, the enzyme responsible for the ring hydroxylation SEYMOUR KAUFMAN droxylation of phenylalanine (17, 18), this product was also shown to be found during the TH-catalyzed conversion of tyrosine to dopa. (For a further discussion of this aspect of the reaction, see Section XI and Fig. 7.)
Intracellular Localization and Regional DistributionAs already mentioned, TH is present in brain, adrenal medulla, and sympathetically innervated tissues. Its intracellular location in these tissues, however, has been the subject of controversy. Udenfriend and co-workers (12) reported that in guinea pig brain all of the activity, and in beef adrenal medulla, most of the activity, was particle bound. They further concluded that the hydroxylase found in the soluble fraction of adrenal medulla was originally present in particles from which it had been released by the homogenization process, a conclusion that was strengthened by the finding that about 90% of the activity was sedimentable after centrifugation for 1 h at 144,000 g. These findings appeared to support the proposal that all of the enzymes involved in catecholamine biosynthesis are localized within a catecholamine-containing granule (19).There is a growing body of evidence that contradicts the conclusion that TH is present intracellularly within a granule (20, 21). For the enzyme from both adrenal medulla and nerve tissue, it has been found that a major fraction of the activity is present in the highspeed supernatant fraction and that the distribution of hydroxylase activity is a function of the composition of the homogenization medium; homogenization in isotonic KC1 leads to more enzyme in the supernatant fraction than does homogenization in isotonic sucrose (21). At least part of the uncertainty about the intracellular localization of TH can be traced to the tendency of the enzyme from bovine adrenal medulla, and probably from other tissues as well, to aggregate and to adsorb to subcellular organelles (21).Although there is now a near consensus in favor of the view that the high molecular weight form of the enzyme in adrenal medulla is an artifact that is formed during cell disruption and that it is not in equilibrium with the native, nonaggregated form (22), that conclusion does not apply to brain where the enzyme appears to exist in two distinct forms, a soluble and a membrane-bound form. In fact, in the earliest studies...
