Fuller B B
Department of Biochemistry and Molecular Biology, University of Oklahoma, Oklahoma City 73190.
In Vitro Cell Dev Biol. 1987 Sep;23(9):633-40. doi: 10.1007/BF02621072.
Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37 degrees C for a minimum of 8 h. Enzyme activity continued to increase for 48 h at which time the maximal level of activation was observed. Activation did not occur at 4 degrees C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation process increased the Vmax of tyrosinase 10-fold and lowered the Km by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole. The "self-activation" response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells.
在37摄氏度孵育至少8小时的Cloudman S - 91小鼠黑色素瘤细胞匀浆中，酪氨酸酶活性增加。酶活性在48小时内持续增加，此时观察到最大激活水平。在4摄氏度时未发生激活，且在细胞的胞质溶胶部分也未发生激活，这表明该反应定位于黑素小体。激活后的酶对非离子去污剂Triton X - 100的增溶作用具有抗性，并且在这种去污剂中制备匀浆不会抑制细胞黑素小体部分的温度依赖性激活。通过酪氨酸羟化酶测定法确定，激活过程使酪氨酸酶的Vmax增加了10倍，Km降低了2倍。酪氨酸酶活性的增加可通过三种测定方法检测到：酪氨酸羟化、黑色素合成以及酪氨酸脱羧。然而，发现黑色素的形成量是酪氨酸羟化或脱羧量的1/20，这一发现表明黑色素途径可能在5,6 - 二羟基吲哚处受阻。用环磷酸腺苷依赖性蛋白激酶孵育细胞匀浆无法模拟“自我激活”反应。添加新鲜细胞提取物可抑制激活后的酪氨酸酶，这一发现表明这些细胞中可能存在酪氨酸酶抑制剂。
