Paul M. Montague

A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase.

The production of melanin pigment in mammals requires tyrosinase, an enzyme which hydroxylates the amino acid tyrosine to DOPA (3,4‐dihydroxyphenylalanine), thus allowing the cascade of reactions necessary to synthesize that biopolymer. However, there are other regulatory steps that follow the action of tyrosinase and modulate the quantity and quality of the melanin produced. DOPAchrome tautomerase is one such melanogenic enzyme that isomerizes the pigmented intermediate DOPAchrome to DHICA (5,6‐dihydroxyindole‐2‐carboxylic acid) rather than to DHI (5,6‐dihydroxyindole), which would be generated spontaneously. This enzyme thus regulates a switch that controls the proportion of carboxylated subunits in the melanin biopolymer. Efforts to clone the gene for tyrosinase have resulted in the isolation of a family of tyrosinase related genes which have significant homology and encode proteins with similar predicted structural characteristics. Using specific antibodies generated against synthetic peptides encoded by unique areas of several of those proteins, we have immuno‐affinity purified them and studied their melanogenic catalytic functions. We now report that TRP‐2 (tyrosinase related protein‐2), which maps to and is mutated at the slaty locus in mice, encodes a protein with DOPAchrome tautomerase activity.

Mammalian tyrosinase: Isozymic forms of the enzyme

Abstract 1. 1. The four isozymes of tyrosinase have been isolated from malignant melanoma tissues and characterized by a variety of biochemical techniques. 2. 2. Electrophoretic and kinetic studies, as well as amino acid analysis, suggest that each isomer has a common subunit requiring only a single genetic locus to determine the enzymes primary structure.

Mammalin tyrosinase. Stoichiometry and measurement of reaction products

The substrates and intermediates involved in the conversion of tyrosine or 3,4-dihydroxyphenylalanine into melanin by autooxidation, or tyrosinases (monophenol, dihydroxyphenylalanine:oxygen oxidoreductases, EC 1.14.18.1) of mushroom or mammalian melanocyte origin, was studied by a variety of enzymic assays, and by amino acid analysis. It was found that the classic pathway of melanin formation was followed, and that the proposed alternate pathway involving formation of the intermediate 3,4,6-trihydroxyphenylalanine was not a functional route, since nascent trihydroxyphenylalanine was not detectable. The ability of isolated mammalian tyrosinases to convert tyrosine into dihydroxyphenylalanine was unequivocably demonstrated. The polymerization of monomers into melanin was followed by the use of specifically labelled precursors, and the data indicate that uncyclized and carboxylated derivatives are not incorporated into the polymer in vitro. It was found that although in most respects the melanin produced from tyrosine by mushroom and mammalian tyrosinses are similar, the control mechanisms involved in the expression of melanin formation in these organisms must differ greatly.

Mammalian tyrosinase. Structural and functional interrelationship of isozymes

The isozymes of tyrosinase from normal and malignant melanocytes were studied; the data indicates that each consists of a basic tyrosinase polypeptide, and differs by post-translational modifications. T3 represents the de novo form of the enzyme; it is converted to T1 in vivo by the addition of sialic acids and neutral sugars, and in turn, to T4 by complexing with mealanosomal membrane constituents. The T2 isomer is suggested to be an artefact of the electrophoretic procedure, and due to deamidation of T3. It is shown that the apparent kinetics of enzyme activity are unafffected by any of these modifications.

Mammalian tyrosinase: isolation by a simple new procedure and characterization of its steric requirements for cofactor activity.

Abstract A method for the isolation of tyrosinase is described, which involves preparative polyacrylamide gel electrophoresis, requires only 24 to 36 h to carry out, and yields ostensibly homogeneous enzyme. The ability of purified tyrosinase to utilize 3,4-dihydroxyphenylalanine (dopa) analogs as cofactors was determined for both of the reactions catalyzed by tyrosinase: (i) tyrosine hydroxylation and (ii) dopa oxidation and melanin formation. The cofactor analogs studied included those in which steric modifying groups were added and those in which substitutions were made at the location of the amine, carboxylic acid, or hydroxyl groups of dopa. The results indicate that each of these groups is essential for maximal enzyme activity and that each is optimally located for tyrosinase activation when in the precise steric conformation found in l -dopa.

Molecular weight estimation of Triton X-100 solubilized proteins by polyacrylamide gel electrophoresis.

Abstract A polyacrylamide gel electrophoresis system is described which employs the nonionic detergent Triton X-100 as a protein solvent. Aside from the obvious advantages of this detergent over ionic detergents and urea in the preservation of protein structure and function, it is demonstrated that the ancillary benefits of gel electrophoresis, i.e., molecular size and charge determination, are possible.

Kinetics of in vitro bovine keratohyalin synthesis

A system is described for the study of the in vitro synthesis of bovine keratohyalin for periods of up to 24 h. Keratohyalin granules appeared morphologically unaltered during culture, although histo‐chetnical stains for RNA indicate a markedly diminished RNA content by 6 h. Incorporation of tritiated histidine began slowly, then became linear between 4 and 24 h in serum‐containing media. However, following pre‐incubation in serum‐free media, increased incorporation occurred from time zero. The internal variation of the system using a standard 6 h pulse was ±10, ±24, ±22, ±18%, in four separate experiments. Measurements of modulations in bovine keratohyalin synthesis in vitro may prove useful in the development of drugs of therapeutic potential in dermatology.