IMPROVED TYROSINASE ACTIVITY STAINS IN POLYACRYLAMIDE ELECTROPHORESIS GELS

Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational processing with possible regulatory implications. So far, SDS-PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However the relatively poor sensitivity of the currently available specific activity stain based on incubation of the gels with L-dopa until the formation of melanin has severely limited the use of electrophoresis in regulation studies. Two alternative staining procedures are presented and discussed. The first one involves the fluorographic detection of radioactive melanin after incubation of the gels in the presence of L-[3-C-14]-dopa. A similar method has already been used by others (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84-89), but its performance has not yet been compared to the one of the dopa procedure. The sensitivity of this method can be varied by adjusting the isotopic dilution of the tracer and/or the time of exposure of the gel, but it is at least ten times higher than the one of the colorimetric stain. Moreover, the intensity of the bands is proportional to the initial tyrosinase activity over a wide range. Using this procedure, the activity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the formation of a colored adduct between dopaquinone and MBTH. Its sensitivity is also more than one order of magnitude higher than the one obtained with L-dopa alone, and comparable to the one of the fluorographic method, but, as opposed to this latter, the complete staining can be performed in less than 1 hr.