Partial characterization of an abundant human skin melanosomal 66 kDa protein (MP 66) and investigation to purify a similar protein from B16 murine melanoma tumours.

A single polypeptide protein of molecular weight 66kDa (MP 66), purified to homogeneity from melanosomes of normal human cadaver skin epidermal melanocytes, was further characterized. Based on the yield in the present investigation, the intracellular concentration of this protein was calculated to be 4.2 microM. It was shown to be a glycoprotein on gel electrophoresis. Based on its partial N-terminal amino acid sequence, it was shown to be distinct from known melanosomal proteins such as gp 75, tyrosinase-related protein-2 (TRP-2) and Pmel 17. Investigation to purify a similar type of protein from B16 murine melanoma tumours by following the same purification procedure resulted in a partially purified protein with a molecular weight of 66 kDa. However, unlike MP 66, this protein did not show inhibition of the monophenolase activity of tyrosinase at pH 6.8. Finally, the effects of 0.5 mM each of CaCl2, ZnSO4 and FeSO4 together, and of human skin epidermal melanosomal proteins, were studied on melanin polymerization at pH 4.7. The metal cations failed to initiate melanin polymerization, while melanosomal proteins did in a dose-dependent manner.

In vitro modulation of proliferation and melanization of melanoma cells by citrate.

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.

Tyrosinase exhibits lag at pH 6.8 under steady state concentrations of tyrosine and 3,4-dihydroxy phenyl alanine in melanoma tissue.

Tyrosine to dopa ratio determines the extent of lag in cresolase activity of tyrosinase when assayed at pH 6.8. The levels of tyrosine and dopa in B-16 murine melanoma tissue were found to be 213 and 13 mmoles/g fresh wt of tissue respectively. Cresolase activity of tyrosinase, when assayed at the above steady state levels of tyrosine and dopa at pH 6.8, exhibited a lag of 5-15 min depending on the amount of enzyme used in the assay mixture and the initial enzyme activity was zero. Under in vivo conditions, the enzyme with zero initial activity can not be active and therefore a far reaching conclusion is that tyrosine to dopa ratio may not regulate the enzyme activity, unlike under in vitro conditions. Possible modes of the regulation of tyrosinase under in vivo conditions are discussed.