Alpha-melanocyte-stimulating hormone regulation of tyrosinase in Cloudman S-91 mouse melanoma cell cultures

Abstract

alpha-MSH (melanocyte-stimulating hormone) causes an increase in tyrosinase activity (O-diphenol-O2 oxidoreductase; EC 1.14.18.1) in Cloudman S-91 mouse melanoma cell cultures following a lag period of approximately 9 h. Treatment of cells with 2 X 10(-7)M alpha-MSH for 6 days results in a 90-fold increase in the specific activity of the enzyme. The hormone-mediated increase in tyrosinase activity is dependent upon continued transcription since the enzyme induction is suppressed by either cordycepin (1 microgram/ml) or alpha-amanitin (10 micrograms/ml). Immunoprecipitation analysis of pulse-labeled tyrosinase from control and MSH-treated cultures (48-h exposure) has demonstrated that MSH stimulates tyrosinase synthesis by approximately 4-fold, a level of induction which does not correspond to the observed 14-fold increase in enzyme activity. When immunotitration curves were developed from cell extracts of control and MSH-treated cultures using immunoprecipitation and competitive enzyme-linked immunosorbent assay protocols, evidence for the presence of immunologically active but catalytically less active enzyme in untreated melanoma cell cultures was demonstrated. Degradation rates of tyrosinase were found to be similar in control cultures or in cells treated with MSH for up to 48 h. Taken together, these results suggest that in addition to stimulating tyrosinase synthesis, MSH may also promote an increase in the catalytic efficiency of the enzyme.