Jara J R, Solano F, Lozano J A
Departamento de Bioquímica, Facultad de Medicina, Universidad de Murcia, Spain.
Pigment Cell Res. 1988;1(5):332-9. doi: 10.1111/j.1600-0749.1988.tb00128.x.
This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.
这项工作描述了一项比较研究，该研究使用三种最广泛使用的方法测定酪氨酸酶活性；两种以L - 酪氨酸为底物的放射性测定法（酪氨酸羟化酶和黑色素形成活性）和一种以L - 多巴为底物的分光光度测定法（多巴氧化酶活性）。这三种方法同时用于测量来自哈丁 - 帕西小鼠黑色素瘤的可溶性、黑素体和微粒体酪氨酸酶同工酶在纯化过程中的活性。本研究的目的是找出三种不同测定法所测酪氨酸酶活性之间的任何相关性，并确定这种相关性是否随同工酶及其纯化程度而变化。结果表明，哺乳动物酪氨酸酶对L - 多巴的转换数比对L - 酪氨酸的转换数更高。因此，以每分钟转化的底物微摩尔数表示的酶活性，在以L - 多巴为底物的测定中比以L - 酪氨酸为底物的测定中更高。此外，羟基化L - 酪氨酸转化为黑色素的百分比很低，并且受几个因素影响，这显然降低了通过黑色素形成测定法测得的酪氨酸酶活性。考虑到这些因素，提出了平均测定间系数。它们的值分别为：将黑色素形成活性转换为酪氨酸羟化酶活性为10，将酪氨酸羟化酶活性转换为多巴氧化酶活性为100，将黑色素形成活性转换为多巴氧化酶活性为1000。还讨论了由于酪氨酸酶制剂中存在抑制剂或黑素生成中与酪氨酸酶无关的调节因子而导致这些值的变化。
