Hamdan F F, Ribeiro P
Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Quebec, Canada.
J Neurochem. 1998 Oct;71(4):1369-80. doi: 10.1046/j.1471-4159.1998.71041369.x.
Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni. We have cloned a novel S. mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Two subsets of SmTH transcripts were identified, one of which carries the S. mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S. mansoni SL. The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa. Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme. Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme. This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation. This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.
儿茶酚胺类物质，如多巴胺和去甲肾上腺素，在包括人体寄生虫曼氏血吸虫在内的所有寄生蠕虫中，作为神经肌肉递质和调节剂发挥着重要作用。我们克隆了一种新的曼氏血吸虫酪氨酸羟化酶（SmTH）cDNA，它与哺乳动物酪氨酸羟化酶具有高度同源性，后者催化儿茶酚胺生物合成的第一步且是限速步骤。鉴定出了SmTH转录本的两个亚组，其中一个在其5'端带有曼氏血吸虫剪接前导序列（SL），而另一个似乎没有与曼氏血吸虫SL进行反式剪接。这两种类型的SmTH转录本编码相同的465个氨基酸的蛋白质，预测大小为54 kDa。SmTH作为N端组氨酸融合蛋白在大肠杆菌中表达产生了一种活性酶，该酶被纯化了约52倍达到表观均一性，最终比活性为0.78微摩尔/分钟/毫克。发现纯化后的酶对四氢生物蝶呤辅因子的绝对需求与哺乳动物酶相同，对高浓度底物酪氨酸抑制的敏感性也相同。纯化后的SmTH也显示出儿茶酚胺产物的特征性抑制作用，尽管对产物抑制的敏感性低于哺乳动物酶。这一证据表明，SmTH编码一种功能性酪氨酸羟化酶，其催化特性与哺乳动物宿主的酶相似，但在调节特性上可能有所不同。在寄生蠕虫中首次证明酪氨酸羟化酶进一步表明，寄生虫具有内源性合成儿茶酚胺的酶能力。
