Regular paper| Volume 2, ISSUE 5, P329-335, September 1991

Effect of DOPA-loading on glutathione metabolising enzymes and tyrosinase in relation to 5-S-cysteinyl-DOPA genesis in cultured B-16 melanoma cells

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      The effect of DOPA and glutathione (GSH) on enzyme systems for 5-S-cysteinyl-DOPA (5SCD) genesis in murine melanoma cells cultured in tyrosine- and cystine-free medium were studied. DOPA at its optimum concentration (10−5 M) when added alone did not alter tyrosinase, glutathione-S-transferase or γ-glutamyl transpeptidase activities. In the presence of GSH at its optimum concentration (10−5 M), DOPA loading did not cause any significant changes in tyrosinase or glutathione-S-transferase (GST) activities. This indicates that the higher 5SCD levels observed in the medium because of DOPA loading in the GSH dependent system results from increased substrate availability rather than the increased enzyme activity. An acute drop in 5SCD at DOPA concentrations above 10−5 M observed in the GSH dependent system may be due to the inhibition of tyrosinase at high substrate concentrations (10−4 M). Conversely, in the presence of DOPA, when GSH was increased, the resultant higher production of 5SCD could be explained by the increased activity of GST. When added alone, GSH (10−5 M) caused a significant increase in GST (≈125%) and γ-GTP (≈50%) activities. A drop in 5SCD in the medium when GSH was added beyond its optimum concentration (10−5 M) in the DOPA-dependent system could be due to competitive inhibition of γ-GTP by GSH.
      The data demonstrate that 5SCD genesis may be enhanced due to the accumulation of cytotoxic melanin precursors such as Math Eq quinone. The relative quantities of GSH at the sites of DOPA quinone formation and the levels of its metabolising enzymes can influence the type of product formed. Although this explains the importance of GST in elevated 5SCD production, the inverse relationship of γ-GTP with 5SCD indicates a complex regulatory influence of melanin precursors on this enzyme in melanoma cells.



      GSH (glutathione), 5SCD (5-S-cysteinyl-DOPA), GST (glutathione-S-transferase), γ-GTP (γ-glutamyl transpeptidase), DQ (DOPA quinone), cys-DOPA (cysteinyl-DOPA), glu-DOPA (glutathionyl-DOPA), GR (glutathione reductase), MEM (minimum essential medium), FCS (fetal calf serum), PBS (phosphate buffered saline), PPB (potassium phosphate buffer), CDNB (1 chloro-2,4 dinitro benzene), ΔE (changes in absorbance)
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