Studies on the Polymorphism of Thiaminase I in Seawater Fish
Studies on the Polymorphism of Thiaminase I in Seawater Fish
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DOI：
10.3177/jnsv.54.339
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发表时间：
2008-10-01
影响因子：
1.6
通讯作者：
Okazaki, Hideki
中科院分区：
文献类型：
--
作者：
Nishimune, Takahiro;Watanabe, Yoshihiro;Okazaki, Hideki
对海水鱼Eisturalia petimba的硫氨酶I进行了表征，首次描述了该酶的pI多态性，并获得了硫氨酶I活性亚片段。E. petimba含有至少三种不同pI的硫胺素酶I，并且主要部分显示pI 5.7。PI同工酶之间最明显的差异是它们解离成的活性亚片段的大小。pI 5.7酶解离成25 kDa的亚片段，而pI 7-9酶解离成约25 kDa的亚片段。22 kDa。与pI 5.7酶相比，在pI 7-9酶中通过吡啶作为第二底物测量的反应速率高3倍。以苯胺为共底物时，不同pI同工酶对镉的抑制程度也有明显差异。当通过疏水性的差异进一步纯化主要pI级分时，得到约100 μ g的较小活性片段。22 kDa的出现，表明同工酶之间的活性亚片段的大小的差异是部分片段化的结果的可能性。将pI 5.7的酶纯化250倍，通过凝胶过滤分析发现最纯化的制剂的大小为106 kDa。纯化的制备物通过SDS-PAGE得到活性25 kDa亚片段，以及15 kDa非活性亚片段。酶是。因此推断其含有活性亚片段以及15 kDa的非活性片段。25 kDa活性亚片段的氨基酸测序显示，与完全加工的N-末端序列一起，两个N-末端肽与额外的Pro-Ser和Gly-Pro-Ser连接，NCBI非冗余数据库没有显示出与其他已知蛋白质的显著相似性。另一方面，来自海水鱼Engraulis japonica内脏的全酶的分子量估计为约1000。100 kDa，通过凝胶过滤色谱法。SDS-PAGE分析表明，它含有一个22 kDa的活性亚片段。
Thiaminase I from the seawater fish Eisturalia petimba was characterized, and pI polymorphism of the enzyme was first described, together with the active subfragment of thiaminase I. The liver of E. petimba contained thiaminase I of at least three different pIs and the major fraction exhibited pI 5.7. The most evident difference among pI isozymes was the size of the active subfragments into which they were dissociated. pI 5.7 enzyme dissociated into subfragments of 25 kDa, while pI 7-9 enzymes dissociated into approx. 22 kDa. The reaction rate measured by pyridine as the second Substrate was three times higher in pI 7-9 enzymes compared with pI 5.7 enzyme. The degree of cadmium inhibition, when aniline was the co-substrate, also showed obvious differences between pI isozymes. When the major pI fraction was further purified by the difference in hydrophobicity, a smaller active fragment of approx. 22 kDa appeared, indicating the possibility that the difference in the size of active subfragment between isozymes is a result of partial fragmentation. The pI 5.7 enzyme was purified 250 times and the size of the Most purified preparation was found to be 106 kDa by gel filtration analysis. The purified preparation gave an active 25 kDa subfragment by SDS-PAGE, together with a 15 kDa non-active subfragment. The enzyme was. thus, inferred to contain active subfragments together with the 15 kDa non-active fragments. Amino acid sequencing of the 25 kDa active subfragment revealed, together with the fully processed N-terminal sequence, two N-terminal peptides with extra Pro-Ser and Gly-Pro-Ser attached to it, and the NCBI non-redundant database did not show significant similarity to other known proteins. On the other hand, the molecular mass of the holoenzyme from the viscera of the seawater fish Engraulis japonica was estimated to be approx. 100 kDa by gel filtration chromatography. An SDS-PAGE analysis revealed that it contained an active subfragment of 22 kDa.