Iwata M, Corn T, Iwata S, Everett M A, Fuller B B
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
J Invest Dermatol. 1990 Jul;95(1):9-15. doi: 10.1111/1523-1747.ep12872677.
Tyrosinase activity was assayed in black and white human foreskin samples by measuring both the hydroxylation of tyrosine to dopa (tyrosine hydroxylase activity) and the conversion of [14C]tyrosine to [14C]melanin (melanin synthesis assay). Enzyme activity was found both in the particulate (75%) and soluble (25%) fractions of the cell. Membrane-bound tyrosinase was readily solubilized by either zwitter-ionic or nonionic detergents. The anionic detergent, sodium cholate, inhibited enzyme activity. Tyrosinase activity in black foreskin homogenates averaged almost three times that in white skin samples (33.8 pmols 3H2O/h/mg skin in black and 12.71 pmoles 3H2O/h/mg skin in white skin), although considerable overlap in activities existed among the two groups. Tyrosinase activities measured with two separate assays, tyrosine hydroxylase and [14C]melanin assays, were similar, suggesting that tyrosine hydroxylase activity is tightly coupled to melanin synthesis. Tyrosinase activity determined by either assay method generally correlated with skin melanin content. Kinetic analysis of tyrosinase from black and white foreskin revealed a Km for tyrosine of 2.5 X 10(-4) M in both skin types. Immunotitration experiments suggested that the difference in tyrosinase activities between white and black skin may be due, not only to different amounts of enzyme present in the melanocytes, but also possibly to differences in the catalytic activities of the enzyme found in melanocytes of black and white skin.
通过测量酪氨酸向多巴的羟基化作用（酪氨酸羟化酶活性）以及[14C]酪氨酸向[14C]黑色素的转化（黑色素合成测定），对黑色和白色人包皮样本中的酪氨酸酶活性进行了测定。在细胞的颗粒部分（75%）和可溶性部分（25%）均发现了酶活性。膜结合酪氨酸酶很容易被两性离子或非离子去污剂溶解。阴离子去污剂胆酸钠会抑制酶活性。黑色包皮匀浆中的酪氨酸酶活性平均几乎是白色皮肤样本的三倍（黑色皮肤为33.8 pmol 3H2O/h/mg皮肤，白色皮肤为12.71 pmol 3H2O/h/mg皮肤），尽管两组之间的活性存在相当大的重叠。用两种单独的测定方法（酪氨酸羟化酶和[14C]黑色素测定）测得的酪氨酸酶活性相似，这表明酪氨酸羟化酶活性与黑色素合成紧密相关。通过任何一种测定方法确定的酪氨酸酶活性通常与皮肤黑色素含量相关。对黑色和白色包皮中酪氨酸酶的动力学分析表明，两种皮肤类型中酪氨酸的Km均为2.5×10(-4)M。免疫滴定实验表明，白色和黑色皮肤之间酪氨酸酶活性的差异可能不仅是由于黑素细胞中存在的酶量不同，还可能是由于黑色和白色皮肤黑素细胞中发现的酶的催化活性不同。
