Russo Gian Luigi, De Nisco Elio, Fiore Gabriella, Di Donato Paola, d'Ischia Marco, Palumbo Anna
Institute of Food Science, National Research Council, Avellino, Italy.
Biochem Biophys Res Commun. 2003 Aug 22;308(2):293-9. doi: 10.1016/s0006-291x(03)01379-2.
The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS-PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.
已证明乌贼（Sepia officinalis）的无黑色素墨水含有一种对多种细胞系（包括PC12细胞）有毒的热不稳定蛋白质成分。凝胶过滤色谱分析表明，有毒成分集中在分子量高于100 kDa且酪氨酸酶活性最高的洗脱组分中。对活性组分进行SDS-PAGE分析显示，有一条主要条带迁移至约100 kDa的分子量处，与无黑色素墨水中单一酪氨酸酶条带的分子量相同。这些数据明确证明了有毒成分与酪氨酸酶的同一性。用固定化蛋白酶K处理纯化的乌贼酪氨酸酶以及蘑菇酪氨酸酶，导致酪氨酸酶活性和细胞毒性同时丧失。乌贼脱辅基酪氨酸酶对PC12细胞无细胞毒性诱导作用。研究发现，纯化的乌贼酪氨酸酶可使PC12细胞中的半胱天冬酶3活性显著增加，最终导致不可逆的凋亡过程。总体而言，这些结果揭示了酪氨酸酶一种迄今未被认识的特性，这可能会促使人们重新评估其生物学意义，而不仅仅将其视为一种单纯的色素生成酶。
