The interaction of acyl-CoA with acyl-CoA binding protein and carnitine palmitoyltransferase I
The interaction of acyl-CoA with acyl-CoA binding protein and carnitine palmitoyltransferase I
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DOI：
10.1016/s1357-2725(01)00049-8
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发表时间：
2001-08-01
影响因子：
4
通讯作者：
Knudsen, J
中科院分区：
文献类型：
--
作者：
Abo-Hashema, KAH;Cake, MH;Knudsen, J
用荧光分析和等温滴定微量热法研究了重组大鼠酰基辅酶A结合蛋白(ACBP)对酰基辅酶A的亲和力，这两种方法都不需要物理分离结合配体和游离配体来确定解离常数(Kd)。11-(丹磺酰氨基)十一酰-辅酶A(Dauda-CoA)从ACBP上的置换得到了竞争酰基-辅酶A的结合参数，与用超灵敏微量热滴定得到的结合参数相比，结果更好。油酰辅酶A和二十二碳六烯基辅酶A的Kd值分别为0.014和0.016微米。在相同的实验条件下，纯化的大鼠肝线粒体的肉碱棕榈酰转移酶I(CPT I)对油酰辅酶A和二十二碳六烯酰辅酶A的K-d值分别为2.4和22.7 PM。考虑到CPT I不仅存在的浓度比ACBP低得多，而且对酰基COAS的亲和力也比ACBP低得多，因此提出CPT I能够直接与ACBP-酰基-CoA二元络合物相互作用。酶的活性与ACBP结合的酰基辅酶A的浓度相关，而与游离酰基辅酶A的浓度无关，这支持了这一事实。酰基辅酶A从ACBP-酰基-辅酶A二元络合物转移到CPT I可能是酶诱导ACBP构象改变导致酰基辅酶A释放的结果。(C)2001爱思唯尔科学有限公司。保留所有权利。
The affinity of recombinant rat acyl-CoA binding protein (ACBP) towards acyl-CoAs was investigated using both fluorimetric analysis and isothermal titration microcalorimetry, neither of which requires the physical separation of bound and free ligand for determining the dissociation constants (Kd). The displacement of 11-(dansyl-amino)undecanoyl-CoA (DAUDA-CoA) from ACBP yielded binding parameters for the competing acyl-CoAs that compared favourably with those obtained using ultra-sensitive micro calorimetric titration. The Kd values of ACBP for oleoyl-CoA and docosahexaenoyl-CoA are 0.014 and 0.016 muM, respectively. Under identical experimental conditions, carnitine palmitoyltransferase I (CPT I) of purified rat liver mitochondria has K-d values of 2.4 and 22.7 PM for oleoyl-CoA and docosahexaenoyl-CoA, respectively. Given that CPT I was not only present at a much lower concentration but also has an appreciably lower affinity for acyl-CoAs than ACBP, it is proposed that CPT I is capable of interacting directly with ACBP-acyl-CoA binary complexes. This is supported by the fact that the enzyme activity correlated with the concentration of ACBP-bound acyl-CoA but not the free acyl-CoA. A transfer of acyl-CoA from ACBP-acyl-CoA binary complexes to CPT I could be a result of the enzyme inducing a conformational alteration in the ACBP leading to the release of acyl-CoA. (C) 2001 Elsevier Science Ltd. All rights reserved.