Comparison of the Catalytic Activities of Three Isozymes of Carnitine Palmitoyltransferase 1 Expressed in COS7 Cells
Comparison of the Catalytic Activities of Three Isozymes of Carnitine Palmitoyltransferase 1 Expressed in COS7 Cells
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DOI：
10.1007/s12010-013-0619-y
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发表时间：
2014-02-01
影响因子：
3
通讯作者：
Shinohara, Yasuo
中科院分区：
文献类型：
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作者：
Hada, Takuya;Yamamoto, Takenori;Shinohara, Yasuo
肉毒碱棕榈酰转移酶1（CPT 1）催化酰基从酰基辅酶A转移到肉毒碱形成酰基肉毒碱，并已鉴定出其三种同工酶1a、1b和1c。有趣的是，据报道1c同工酶没有显示出酶活性，但并没有清楚地证明这种失活是由于其功能障碍还是由于其表达不良。在本研究中，我们（a）在COS 7细胞中表达了单个CPT 1同工酶，（B）使用三种细菌表达的CPT 1同工酶作为标准，通过Western印迹定量评价了它们的表达水平，以及（c）评价了它们的催化活性。通过这些实验，我们成功地证明了1c同工酶的酶活性的缺乏是由于其功能障碍。此外，标准CPT 1同工酶的制备实验表明，1c同工酶在SDS-PAGE凝胶中的迁移与分子大小之间没有标准关系。我们进一步试图通过制备1a和1c之间的嵌合CPT 1来确定1c同工酶为什么是惰性的，但由于嵌合CPT 1之一没有充分表达，因此无法得出明确的结论。
The enzyme carnitine palmitoyltransferase 1 (CPT1) catalyzes the transfer of an acyl group from acyl-CoA to carnitine to form acylcarnitine, and three isozymes of it, 1a, 1b, and 1c, have been identified. Interestingly, the 1c isozyme was reported to show no enzymatic activity, but it was not clearly demonstrated whether this inactivity was due to its dysfunction or due to its poor expression. In the present study, we (a) expressed individual CPT1 isozymes in COS7 cells, (b) evaluated quantitatively their expression levels by Western blotting using the three bacterially expressed CPT1 isozymes as standards, and (c) evaluated their catalytic activities. With these experiments, we successfully demonstrated that the absence of the enzymatic activity of the 1c isozyme was due to its dysfunction. In addition, experiments on the preparation of standard CPT1 isozymes revealed that the 1c isozyme did not show the standard relationship between migration in an SDS-PAGE gel and molecular size. We further tried to determine why the 1c isozyme was inert by preparing chimeric CPT1 between 1a and 1c, but no clear conclusion could be drawn because one of the chimeric CPT1s was not sufficiently expressed.