Tyrosinase isoenzymes in mammalian melanocytes

Abstract

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...

Melatonin Antagonizes α‐Melanocyte‐Stimulating Hormone Enhancement of Melanogenesis in Mouse Melanoma Cells by Blocking the Hormone‐Induced Accumulation of the C Locus Tyrosinase

“…Conversely, a-MSH mediates the preferential induction of a tyrosinase isoenzyme of higher electrophoretic mobility, termed HEMT (Valverde et al, 1993). This form of tyrosinase displays higher tyrosine hydroxylase and dopa oxidase specific activities (JimCnez-Cervantes et al, 1993b, 1994.…”

“…It has been clearly shown that both continued transcription and translation are necessary for a-MSH and theophylline (Valverde et al, 1993) stimulation of tyrosinase activity in mouse melanoma cells. Therefore, extracellular signals interfering with transcription of the tyrosinase gene and/or with translation of the corresponding mRNA should antagonize this stimulation.…”

Isoliquiritigenin-Induced Differentiation in Mouse Melanoma B16F0 Cell Line

“…Morphological changes with dendrite outgrowth and melanogenesis are considered specific differentiation markers for B16F0 cells [24]. TYR activity and melanin content are well-known molecular markers of melanoma cellular differentiation [25, 26]. TYR is very important in controlling melanogenesis.…”

Tyrosinase Isoenzymes: Two Melanosomal Tyrosinases With Different Kinetic Properties and Susceptibility to Inhibition by Calcium

“…These forms, termed LEMT (Low electrophoretic mobility tyrosinase) and HEMT (high electrophoretic mobility tyrosinase) cannot be interconverted by either limited proteolysis or deglycosylation and can be partially resolved by differential solubilization with the non ionic detergent Brij 35 (JimBnez-Cervantes et al, 1993a). Moreover, they are stimulated to different degrees by aMSH or theophylline treatment of B16 melanocytes in culture (Valverde et al, 1993). Therefore, these tyrosinases could be different isoenzymes rather than isoforms.…”

“…Therefore, the regulatory role of calcium appears to be complex and may involve various effects at different steps of the melanogenesis pathway. The interpretation of these data is further complicated since the effects of calcium on purified tyrosinase have not yet been adequately characterized, and since the tyrosinase activity in basal and MSH stimulated conditions may correspond to two different isoenzymes with distinct kinetic properties and structure (Valverde et al, 1993). Therefore, we tested the effect of calcium at physiologically relevant concentrations on purified tyrosinase isoenzymes.…”