IMPROVED TYROSINASE ACTIVITY STAINS IN POLYACRYLAMIDE ELECTROPHORESISGELS

Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational proces sing with possible regulatory implications. So far, SDS-PAGE has prove n to be the method of choice for the resolution of tyrosinase isoforms . However the relatively poor sensitivity of the currently available s pecific activity stain based on incubation of the gels with L-dopa unt il the formation of melanin has severely limited the use of electropho resis in regulation studies. Two alternative staining procedures are p resented and discussed. The first one involves the fluorographic detec tion of radioactive melanin after incubation of the gels in the presen ce of L-[3-C-14]-dopa. A similar method has already been used by other s (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84-89), but it s performance has not yet been compared to the one of the dopa procedu re. The sensitivity of this method can be varied by adjusting the isot opic dilution of the tracer and/or the time of exposure of the gel, bu t it is at least ten times higher than the one of the colorimetric sta in. Moreover, the intensity of the bands is proportional to the initia l tyrosinase activity over a wide range. Using this procedure, the act ivity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the for mation of a colored adduct between dopaquinone and MBTH. Its sensitivi ty is also more than one order of magnitude higher than the one obtain ed with L-dopa alone, and comparable to the one of the fluorographic m ethod, but, as opposed to this latter, the complete staining can be pe rformed in less than 1 hr.