Tyrosinase in rat liver and brain

Presence of tyrosinase (EC 1.14.18.1), the enzyme that produces quinone-free radicals and therefore has a potential for tissue damage, is demonstrated in normal rat liver and brain. The enzyme displays both L-DOPA oxidase and tyrosine hydroxylase activities. It is associated with the mitochondrial and nuclear membranes and occurs in multiple forms, only one which is the same in both subcellular locations. Up to the present time tyrosinase was thought to occur only in melanomas and melanogenic tissues.

Tyrosine hydroxylase activity was assayed as described by Hearing and Ekel (1) with some minot modifications. L-DOPA-oxidase activity measuring the production of labelled melanin using 14C-L-DOPA was assayed and counted as described by Hearing (2). Protein was determined using the Lowry procedure (3) with bovine serum albumin as the standard. Dopamine oxidase activity was measured as described for L-DOPA oxidase activity except 1 mM dopamine. (8-14C dopamine, sp. Act. -0.5 mCi/nmol, Amersham) + 0.1mM Deprenyl and 0.1mM Clorgyline were used instead of 1 mM L-DOPA.

Monoamine oxidase was assayed as described by Heikkela et al. (4) an aldehyde dehydrogenase was assayed as described by Greenfield and Pietruszko (5). Subcellular fractionation was performed on the liver and brain of adult female Sprague-Dawley rats, 80-120 days old from Charles River. All subcellular fractions consisting of cytoplasm, mitochondria, microsomes and nuclei were separated via a procedure of Hogeboom (6). The nuclear fraction was pelleted via centrifugation at 600 x g for 10 minutes at 4C in a Sorvall RC-58 ultracentrifuge. The nuclei were then purified from plasma membranes by homogenization and using a two-step sucrose gradient according to the procedure of Fleischer and Kervina (7).

Incubation of intact rat liver mitochondria with dopamine (1 mM) for 90-120 minutes at 37C resulted in formation of melanin. In the absence of rat liver mitochondria or in the presence of bovine serum albumin there was no melanin, suggesting that melanin formation was catalyzed by an enzyme. The suspected enzyme was monoamine oxidase; however, incubations of rat liver mitochondria with dopamine in the presence of specific monoamine oxidase inhibitors Deprenyl (0.1 mM) and/or Clorgyline (0.1 mM) (separately and together) failed to abolish melanin formation. When, instead of dopamine, 1 mM L-DOPA (which is not a substrate for monoamine oxidase) was used, melanin formation also occurred. This suggested another enzyme capable of forming melanin from dopamine or L-DOPA. When liver mitochondrial membranes were incubated with1mM L-DOPA and various concentrations of 1-phenyl-2-thiourea (an inhibitor of tyrosinase) 1 mM, 0.5 mM and 0.1mM at 37C, the formation of melanin was inhibited; the inhibition was proportional to the 1-phenyl-2-thiourea concentration.

Employing subcellular fractionation procedures combined with the activity assays, tyrosinase was localized in the mitochondrial and nuclear membranes of rat liver: it was also found in the mitochondrial membranes from rat brain (Table 1).

The time courses of DOPA and dopamine oxidation were determined with intact liver mitochondria and with mitochondrial membranes. When intact mitochondria were used, melanin formation was preceeded by a 90 min. lag phase, this lag completely disappeared when purified mitochondrial membranes were used, suggesting presence of tyrosinase inhibitors in the intact mitochondria. The enzyme from liver mitochondrial and nuclear membranes was solubilized with sodium deoxycholate and subjected to isoelectric focusing: seven activity bands were observed in nuclear membranes and only three bands were seen in the mitochondrial membranes, only one band (pI =5.1) was common to both organelles.

Tyrosinase is a potentially dangerous enzyme because it produces quinone-free radicals from endogenous amines. These radicals are extremely reactive and can readily form adducts with proteins, they also react with each other to produce melanin. When rat liver mitochondria are incubated with 14C-dopamine or DOPA, binding of radioactivity to the mitochondrial protein occurs with the concurrent loss of mitochondrial matrix aldehyde dehydrogenase activity.

This also occurs when rat mitochondrial membranes and human aldehyde dehydrogenase (E2) are incubated with dopamine. The activity loss is associated with melanin formation and occurs in the absence of monoamine oxidase activity.

Inactivation of aldehyde dehydrogenase is associated with incorporation of label from 14C-dopamine which has been visualized via SDS gel electrophoresis of the mitochondrial matrix proteins followed by Western blotting and autoradiography. A protein of MW of 54,000 which also reacted with the antibody against human mitochondrial aldehyde dehydrogenase, was found to incorporate radioactivity from 14C-labelled dopamine. Two other proteins (identity unknown) with subunit MW of ca. 20,000 and 120,000 also incorporated the label. Since aldehyde dehydrogenase inactivation simultaneously occurs with melanin formation in the absence of monoamine oxidase activity it appears that tyrosinase is the enzyme that inactivates aldehyde dehydrogenase; the reaction leading to inactivation could occur via o-quinones.

References
1. Hearing VJ, Ekel TM (1976) Biochem J 157:549-557.
2. Hearing VJ (1987) in : Methods in Enzymology (Kaufman S et al, eds), Vol. 142, pp. 154-165, Academic Press.
3. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193:265-275.
4. Heikkila RE, Manzino L, Cabbat FS, Randall RJ (1985) J Neurochem 45:1049-1054.
5. Greenfield NJ, Pietruszko R (1977) Biochem Biophys Acta 483:35-45.
6. Hogeboom GD (1955) Methods Enzymol 1:16-19.
7. Fleischer S, Kervina M (1974) in : Methods in Enzymology (Colowick SP, Kaplan NO, eds), Vol. 31, pp 6-41, Academic Press.

Table 1

Tyrosinase activity of subcellular fractions of rat liver and brain

Subcellular fractions Activity (pmol/min/mg protein)

Liver
Mitochondrial membranes
Nuclear membranes
Nuclear soluble

Plasma membranes

Brain
Mitochondrial membranes
Nuclear membranes


Liver
Mitochondrial membranes

(a) 14C L-DOPA oxidase activity

5.4
2.8
none

none


2.1
0.39

(b) 3H-tyrosine hydroxylase activity

3.2 x 10-3

 

SF Turan and R. Pietruszko
Center of Alcohol Studies
Rutgers University
Piscataway, New Jersey 08855-0969, USA