大鼠肝脏线粒体酰基辅酶A合成酶的分光光度法研究。

Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria.

作者信息

Garland P B, Yates D W, Haddock B A

出版信息

Biochem J. 1970 Sep;119(3):553-64. doi: 10.1042/bj1190553.

DOI:10.1042/bj1190553

PMID:5500316

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1179387/

Abstract

Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.-) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with epsilon(mM) 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg(2+) and corresponded to a previously described ;external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg(2+). Of these two ;internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.-), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases.

摘要

癸-2,4,6,8-四烯酸是大鼠肝脏线粒体中ATP特异性（EC 6.2.1.2或3）和GTP特异性（EC 6.2.1.-）酰基辅酶A合成酶的底物。癸四烯酰辅酶A的酶促合成产生了新的光谱特征。酰基辅酶A减去游离酸的差示光谱在376nm处有最大值，ε（毫摩尔）为34。等吸收点在345nm和440nm处。2. 线粒体悬浮液中癸四烯酸对辅酶A的酰化作用可用双波长分光光度计连续测定。3. 利用该技术，在大鼠肝脏线粒体中证明了三种不同类型的酰基辅酶A合成酶活性。其中一种利用添加的辅酶A和ATP，需要添加Mg(2+)，对应于先前描述的“外部”酰基辅酶A合成酶。另外两种酰基辅酶A合成酶活性利用线粒体内的辅酶A，不需要添加Mg(2+)。在这两种“内部”酰基辅酶A合成酶中，一种对解偶联剂不敏感，受磷酸盐或砷酸盐抑制，对应于GTP特异性酶。另一种对应于ATP特异性酶。4. 仅当能量来源为添加的ATP时，白术酸才抑制两种内部酰基辅酶A合成酶的活性。5. 癸四烯酸酰化的线粒体内辅酶A的量与内部ATP特异性或GTP特异性酰基辅酶A合成酶是否活跃无关。得出的结论是，这两种内部酰基辅酶A合成酶可利用相同的线粒体内辅酶A池。6. 添加棕榈酰-dl-肉碱、2-氧代戊二酸或丙酮酸会减少可被癸四烯酸酰化的线粒体内辅酶A的量。这些观察结果表明，丙酮酸脱氢酶（EC 1.2.4.1）、氧代戊二酸脱氢酶（EC 1.2.4.2）、肉碱棕榈酰转移酶（EC 2.3.1.-）、柠檬酸合酶（EC 4.1.3.7）和琥珀酰辅酶A合成酶（EC 6.2.1.4）都与两种内部酰基辅酶A合成酶一样，可利用相同的线粒体内辅酶A池。