Effect of heat treatment on proper oligomeric structure formation of thermostable glutamate dehydrogenase from a hyperthermophilic archaeon.
Effect of heat treatment on proper oligomeric structure formation of thermostable glutamate dehydrogenase from a hyperthermophilic archaeon.
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热处理对来自超嗜热古菌的热稳定谷氨酸脱氢酶的适当寡聚结构形成的影响。
DOI：
10.1006/bbrc.1997.7850
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发表时间：
1997
影响因子：
3.1
通讯作者：
T. Imanaka
中科院分区：
文献类型：
--
作者：
R. N. Abd Rahman;S. Fujiwara;M. Takagi;S. Kanaya;T. Imanaka
从超嗜热古菌Pyrococcus sp.KOD1中纯化天然谷氨酸脱氢酶（Pk-GDH），并与大肠杆菌中表达的重组酶进行活性和结构比较。通过SDS-PAGE和凝胶过滤测定这些酶的分子量揭示，天然酶仅作为六聚体形式被纯化，而重组酶作为单体和六聚体形式被纯化。酶活性的测定表明，只有六聚体形式的酶是有活性的。此外，注意到重组酶的六聚体形式的比活性远低于天然酶的比活性，并且这些酶的圆二色谱彼此明显不同。这些结果表明，具有低比活性的重组酶（I型）的六聚体形式的结构不同于具有高比活性的天然酶（II型）的结构。在热处理（80 ℃，15分钟）后，I型结构有效地转化为II型结构，并且酶的比活性增加了2.6倍。同样地，在热处理（70 ℃，15分钟）后，重组酶的无活性单体形式至少部分地与六聚体形式结合。这些结果表明，高温对PK-GDH的正确折叠和寡聚化起着重要作用。
Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli. Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold. Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.
影响因子：
3.9
作者：
Xie, ZJ;Wang, YH;Askari, A
通讯作者：
Askari, A
从折叠单体组装天冬氨酸转氨甲酰酶催化三聚体。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Burns,DL;Schachman,HK
通讯作者：
Schachman,HK