Department of Physiology, Eberhard-Karls-University of Tuebingen, Gmelinstr. 5, 72076 Tübingen, Germany.
Toxins (Basel). 2014 Mar 18;6(3):1096-108. doi: 10.3390/toxins6031096.
Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis.
Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry.
A 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca2+]i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2+. Tyrosinase did not significantly modify forward scatter.
Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca2+ and ceramide formation.
蘑菇酪氨酸酶是一种含铜酶，可修饰肿瘤细胞的生长和存活。蘑菇酪氨酸酶可能促进细胞凋亡，这种作用部分归因于对线粒体功能的干扰。红细胞缺乏线粒体，但能够发生类似于细胞凋亡的自杀性细胞死亡或红细胞皱缩，其特征是细胞体积缩小和细胞膜混乱，导致磷脂酰丝氨酸在红细胞表面暴露。参与触发红细胞皱缩的信号包括细胞质 Ca2+ 活性 ([Ca2+]i) 的增加和鞘磷脂酶的激活，随后形成神经酰胺。本研究探讨了酪氨酸酶是否刺激红细胞皱缩。
通过流式细胞术，从正向散射估计细胞体积，从膜联蛋白 V 结合估计磷脂酰丝氨酸暴露，从 Fluo3-荧光估计 [Ca2+]i，从荧光抗体结合估计神经酰胺丰度。
蘑菇酪氨酸酶（7 U/mL）暴露 24 小时后，[Ca2+]i 显著增加，神经酰胺丰度显著增加，膜联蛋白 V 结合显著增加。在不存在细胞外 Ca2+的情况下，酪氨酸酶处理后的膜联蛋白 V 结合显著减弱，但并未完全阻断。酪氨酸酶对前向散射没有显著影响。
酪氨酸酶触发细胞膜混乱，这种作用至少部分归因于细胞外 Ca2+的进入和神经酰胺的形成。
