Chaves L B, Briones M R, Schenkman S
Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, São Paulo, Brazil.
Mol Biochem Parasitol. 1993 Sep;61(1):97-106. doi: 10.1016/0166-6851(93)90162-q.
We have studied the trans-sialidase from insect forms of Trypanosoma cruzi growing in axenic culture. Log phase epimastigotes expressed little or no trans-sialidase activity, and were unable to incorporate exogenous sialic acid. Transsalidase started to be expressed at the late logarithmic phase, with specific activity increasing steadily as the culture reached the stationary phase. Trans-sialidase was purified from the late log phase epimastigote culture, which contained less than 2% of metacyclic forms, yielding a glycoprotein that migrated as a single 90-kDa band in sodium dodecyl sulfate gels. This enzyme features: (1) no reaction with antibodies against the peptide repeats present in the carboxy-terminal of trypomastigote trans-sialidase; (2) positive reaction with antibodies raised against a fragment of trypomastigote trans-sialidase that contains the active site; (3) similar kinetic properties and identical acceptor-donor specificity when compared to the trypomastigote enzyme; and (4) neuraminidase activity in the absence of acceptors. Upon differentiation into metacyclic forms, a trans-sialidase activity containing the carboxy-terminal repeats of the trypomastigote enzyme was released into the medium. These results suggest that epimastigotes express a developmentally regulated trans-sialidase that contains the same catalytic site but lacks the tandem amino acid repeats typical of trypomastigote trans-sialidase.
我们研究了在无细胞培养中生长的克氏锥虫昆虫型的转唾液酸酶。对数期的上鞭毛体几乎不表达或不表达转唾液酸酶活性，并且无法摄取外源性唾液酸。转唾液酸酶在对数后期开始表达，随着培养物进入稳定期，其比活性稳步增加。转唾液酸酶是从对数后期的上鞭毛体培养物中纯化得到的，该培养物中含有不到2%的后循环型，得到一种糖蛋白，在十二烷基硫酸钠凝胶中迁移为单一的90 kDa条带。这种酶具有以下特点：(1) 与抗锥鞭毛体转唾液酸酶羧基末端肽重复序列的抗体无反应；(2) 与针对包含活性位点的锥鞭毛体转唾液酸酶片段产生的抗体呈阳性反应；(3) 与锥鞭毛体酶相比，具有相似的动力学性质和相同的受体-供体特异性；(4) 在没有受体的情况下具有神经氨酸酶活性。分化为后循环型时，含有锥鞭毛体酶羧基末端重复序列的转唾液酸酶活性释放到培养基中。这些结果表明，上鞭毛体表达一种受发育调控的转唾液酸酶，其含有相同的催化位点，但缺乏锥鞭毛体转唾液酸酶典型的串联氨基酸重复序列。
