A method for detecting dopaminechrome isomerase activity on gels.

Melanogenesis involves oxidation of 3,4-dihydroxyphenylalanine (dopa) to dopachrome which then is converted into 5,6-dihydroxyindole by dopachrome isomerase. 5,6-Dihydroxyindole is oxidized to its quinone which in turn is metabolized nonenzymatically to melanin. In addition to dopachrome isomerase, a new dopminechrome isomerase activity involved in the conversion of dopaminechrome into 5,6-dihydroxyindole has been observed in the larva of Rhinoceros oryctes. This dopaminechrome isomerase differs from dopachrome isomerase in its electrophoretic mobility and substrate specificity. The present study reports a specific, sensitive and rapid staining method for detecting dopaminechrome isomerase activity after electrophoresis. Using this new method, the presence of the dopaminechrome isomerase activity, which is involved in melanogenesis, could easily be detected by staining tyrosinase embedded native gels in dopamine solution. Tyrosinase entrapped in the gels converts dopamine in dopaminechrome. The dopaminechrome isomerase separated in the gels catalyzes dopaminechrome to 5,6-dihydroxyindole which is oxidized further by tyrosinase to colored melanochrome. The dopaminechrome isomerase appears as a bluish purple band against a pink background.

[1]  C. Amaratunga,et al.  Complex formation between mushroom tyrosinase and Manduca dopachrome isomerase. , 1995, Pigment cell research.

[2]  K. Nellaiappan,et al.  Detection of dopachrome isomerase activity on gels. , 1994, Analytical biochemistry.

[3]  K. Valivittan,et al.  Differentiation of tyrosine hydroxylase and phenol oxidase after electrophoresis. , 1993, Biotechnic & histochemistry : official publication of the Biological Stain Commission.

[4]  L. Riddiford,et al.  Molecular mechanisms of cuticular melanization in the tobacco hornworm, Manduca sexta (L.) (Lepidoptera : Sphingidae) , 1993 .

[5]  Francisco J. Silva,et al.  Catecholamines in drosophila melanogaster: DOPA and dopamine accumulation during development , 1992 .

[6]  M. Sugumaran,et al.  Quinone methide as a new intermediate in eumelanin biosynthesis. , 1991, The Journal of biological chemistry.

[7]  J. Pawelek.  Dopachrome conversion factor functions as an isomerase. , 1990, Biochemical and biophysical research communications.

[8]  K. Nellaiappan,et al.  On the latency and nature of phenoloxidase present in the left colleterial gland of the cockroach Periplaneta americana. , 1990, Archives of insect biochemistry and physiology.

[9]  D. Townsend,et al.  Function of dopachrome oxidoreductase and metal ions in dopachrome conversion in the eumelanin pathway. , 1988, Biochemistry.

[10]  S. Tojo,et al.  Hormonal control of cuticular melanization in the common cutworm, Spodoptera litura , 1988 .

[11]  K. Kramer,et al.  Cuticular strength and pigmentation of rust-red and black strains of Tribolium castaneum: Correlation with catecholamine and β-alanine content , 1987 .

[12]  T. Wright.  The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. , 1987, Advances in genetics.

[13]  P. Gettins,et al.  Synthesis in vitro of 5,6-dihydroxyindole-2-carboxylic acid by dopachrome conversion factor from Cloudman S91 melanoma cells. , 1985, The Journal of investigative dermatology.

[14]  K. Kramer,et al.  Properties of tyrosinase and dopa quinone imine conversion factor from pharate pupal cuticle of Manduca sexta L. , 1984 .

[15]  K. Kramer,et al.  N-β-Alanyldopamine: Major Role in Insect Cuticle Tanning , 1982, Science.

[16]  H. Yamazaki.  The cuticular phenoloxidase in Drosophila virilis , 1969 .

[17]  N. M. Summers.  Cuticle sclerotization and blood phenol oxidase in the fiddler crab, Uca pugnax. , 1967, Comparative biochemistry and physiology.

[18]  P. Karlson,et al.  Zum tyrosinstoffwechsel der insekten IX. Kontrolle des tyrosinstoffwechsels durch ecdyson , 1962 .

[19]  H. S. Mason.  The chemistry of melanin; mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. , 1948, The Journal of biological chemistry.