E. Benedito et al., MELANIN FORMATION IN THE INNER-EAR IS CATALYZED BY A NEW TYROSINE-HYDROXYLASE KINETICALLY AND STRUCTURALLY DIFFERENT FROM TYROSINASE, Biochimica et biophysica acta (G). General subjects, 1336(1), 1997, pp. 59-72
Detergent solubilized extracts of the cochleae of adult gerbils (Merio
nes unguiculatus) contain a tyrosine hydroxylase activity measurable b
y the radiometric method of Pomerantz. This activity is not related to
Fenton-type reactions, since it is not inhibited by free radical scav
engers and is heat and protease sensitive. It does not appear to be re
lated to a peroxidase (EC 1.11.1.7) since it is neither dependent on H
2O2, nor inhibited by catalase (EC 1.11.1.6). The involvement of a tyr
osine hydroxylase (EC 1.14.16.2) related to catecholamine synthesis is
also unlikely, since the activity is highly sensitive to 2-mercaptoet
hanol and is not increased by addition of tetrahydrobiopterin. The act
ivity in crude inner ear extracts displayed an unusual maturation beha
viour, with a slow activation upon aging at 4 degrees C. Fully active
enzyme displayed Michaelis-Menten kinetics, with a K-m for L-tyrosine
of 47 mu M. Cochlear tyrosine hydroxylase, but not melanoma tyrosinase
(EC 1.14.18.1), was inhibited by o-phenanthroline, and was not depend
ent on L-DOPA as cofactor for full enzymatic activity. Crude extracts
were also able to catalyze L-DOPA oxidation and melanin formation from
either L-tyrosine or L-DOPA. The tyrosine hydroxylase, DOPA oxidase a
nd melanin formation activities most probably resided in the same mole
cule, as suggested by inhibition studies. A tyrosine hydroxylase and m
elanin formation activity with identical properties was found in prima
ry cultures of stria vascularis melanocytes. Immunochemical evidence c
onfirmed the absence of either the tyrosinase encoded for by the albin
o locus, or the tyrosinase isoenzyme TRP1, encoded for by the brown lo
cus. Conversely, an immunorreactive band of molecular weight 70 kDa wa
s specifically recognized by a tyrosinase polyclonal antiserum in West
ern blot experiments. These results prove that melanogenesis in the co
chlea, and likely in other extracutaneous locations such as the brain,
is catalyzed by enzymatic systems different from, but related to tyro
sinase. (C) 1997 Elsevier Science B.V.