Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells Evidence for a differential induction of two distinct isoenzymes

Valverde, Paloma; García-Borrón, J.C.; Martinez-Liarte, J. H.; Solano, Francisco; Lozano, José Antonio

doi:10.1016/0014-5793(92)80600-l

Cited by 12 publications

(11 citation statements)

References 15 publications

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“…In fact we have previously found that there is no kinetic evidence suggesting that a modulation of CAMP levels might result in either the removal of an inhibitor or the induction of an activator [17]. However, our results d o not exclude the possibility that such effectors could indeed exist and contribute to the control of tyrosinase activity by other signal transduction pathways such as protein kinase C activation [33, 341 or …”

Section: Discussioncontrasting

confidence: 65%

Tyrosinase isoenzymes in mammalian melanocytes

Valverde

García‐Borrón

Jiménez‐Cervantes

et al. 1993

European Journal of Biochemistry

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In mouse melanoma melanocytes, a-melanocyte-stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH, or its superpotent analog [Ahx4, ~P h e~l M s H promotes a larger induction of tyrosine hydroxylase than of dopa oxidase activity in whole cell extracts. This higher activation of tyrosine hydroxylation was found not only in the melanosomal but also in the microsomal fraction; it appears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase activity of control and theophylline-treated extracts displayed several kinetic differences, including different K,, values for both substrates and requirements for the cofactor L-dopa. SDSPAGE, followed by a sensitive specific activity stain, demonstrated that melanosomes of control cells contain one lower-electrophoretic-mobility form of tyrosinase, whereas melanosomes of cells treated with either theophylline or MSH display, in addition to the lower-mobility form, a faster-migrating activity band. These tyrosinase forms are not interconvertible by proteolysis or deglycosylation. Their nature is discussed as related to the properties of the previously described low-and high-electrophoretic-mobility tyrosinases (LEMT and HEMT), as well as of the proteins encoded by the c and b loci.Tyrosinase (monophenol monooxygenase) is the enzyme controling the rate-limiting step in melanin biosynthesis, the hydroxylation of L-tyrosine to 3,4-dihydroxy-~-phenylalanine (1-dopa) [l, 21. A variety of mammalian melanocytes in culture have been shown to respond to treatment with a-melanocyte-stimulating hormone (MSH) with large increases in tyrosinase activity, leading ultimately to a visible increase in the amount of pigment formed within the cells Enzyme. Tyrosinase, monophenol monooxygenase (EC 1.14.18.1).161 are able to mimick the hormone action, and even to achieve larger activations of the enzyme. However, the molecular mechanisms coupling the increase in intracellular cAMP to tyrosinase activation are unknown, and several possible explanations have been proposed. This increase might just reflect an increased tyrosinase abundance within stimulated cells [16, 181, or might result from two complementary processes, one leading to an increase in tyrosinase abundance and the other to the activation of a pre-existing pool of inactive tyrosinase molecules [9, 19, 201. According to this last hypothesis, only moderate increases in tyrosinase mRNA levels [21, 221 and in the rates of synthesis and degradation of the protein [9, 201 have been detected in MSHstimulated cells. However, continued transcription and translation has been reported to be essential for tyrosinase activation [9].On the other hand, it has been shown that mouse, and possibly human, melanocytes contain two tyr...

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Section: Discussioncontrasting

confidence: 65%

Tyrosinase isoenzymes in mammalian melanocytes

Valverde

García‐Borrón

Jiménez‐Cervantes

et al. 1993

European Journal of Biochemistry

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“…100% values were: CV: 6,000,000 cells; MC: 3.45 pg/ml; DO: 4.08 mU/mg; DCT: 3.78 mU/mg. true tyrosinase and TRP1, are encoded by the albino and brown loci respectively (Jimenez et al, 1991), and they have different ratio of tyrosine hydroxylase to dopa oxidase activities (Valverde et al, 1992). DCT has been recently shown to be identical to TRP2, the protein encoded by the slaty locus (Tsukamoto et al, 1992).…”

Section: Results and Discussion Effect Of Fungizone Concentrationmentioning

confidence: 99%

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

Peinado

Martinez-Liarte

Solano

et al. 1992

Pigment Cell Research

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The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.

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“…proteins encoded by the same gene but with different post-translational modifications, or rather tyrosinase isoenzymes encoded by different genes remains unsettled. This question has important biological implications, since it has been reported that the expression of the b and c proteins is different in malignant and normal melanocytes [20] and since it has been recently proposed that B16 mouse malignant melanocytes could indeed contain two different isoenzymes with different responses to rx-melanocyte-stimulating hormone [26].…”

mentioning

confidence: 99%

Tyrosinase isoenzymes in mammalian melanocytes

Jiménez‐Cervantes

García‐Borrón

Valverde

et al. 1993

European Journal of Biochemistry

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B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDSPAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane andor the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Ktn for 3,4-dihydroxy-~-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of K,, for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L-dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2-phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.Mammalian tyrosinase (monophenol monooxygenase) is a copper-containing glycoprotein responsible for the synthesis of melanin within melanocytes [l, 21. The enzyme catalyses the rate-limiting step in melanin biosynthesis, the hydroxylation of L-tyrosine to 3,4-dihydroxy-~-phenylalanine @-dopa) and the subsequent oxidation of L-dopa to L-dopaquinone. In the absence of thiol compounds L-dopaquinone undergoes a rapid oxidation and spontaneous rearrangement leading to L-dopachrome, and ultimately, to the melanin polymer [3]. Although melanin formation can proceed spontaneously from L-dopaquinone, at least one other enzyme involved in the process has been characterized [4-61. This enzyme, called dopachrome tautomerase [5], catalyses the non-decarboxylative rearrangement of L-dopachrome, yielding 5,6-dihydroxyindole 2-carboxylic acid. In the absence of the enzyme, L-dopachrome spontaneously evolves to 5,6-dihydroxyindole. The series of oxidation and polymerization reactions leading from 5,6-dihydroxyindole 2-carboxylic acid and 5,6-dihydroxyindole to melanin is still poorly understood, although it has been shown that 5,6-dihydroxyindole is essential for the in vitro polymerization of its 2-carboxylic acid [7], and that tyrosinase might play a role in this distal phase of melanogenesis [7, 81.

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Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells Evidence for a differential induction of two distinct isoenzymes

Cited by 12 publications

References 15 publications

Tyrosinase isoenzymes in mammalian melanocytes

Tyrosinase isoenzymes in mammalian melanocytes

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

Tyrosinase isoenzymes in mammalian melanocytes

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