McMillin J B, Wang D, Witters L A, Buja L M
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Medical School at Houston 77030.
Arch Biochem Biophys. 1994 Aug 1;312(2):375-84. doi: 10.1006/abbi.1994.1322.
An understanding of the mechanism of malonyl-CoA interaction with carnitine palmitoyltransferase (CPT-I) in isolated mitochondria is complicated by membrane fragmentation and CPT-II exposure. Using cultured neonatal rat cardiac myocytes, as in situ model was developed to measure CPT-I. In the cardiac cells treated with 5 microM digitonin, CPT-II contamination of CPT activity is 0.62% as quantitated by citrate synthase activity present in damaged myocytes under assay conditions. Moreover, the sensitivity of myocyte CPT-I to malonyl-CoA, its substrate preference for decanoyl-CoA and the affinity of CPT-I for l-carnitine (0.19 mM) are comparable with similar measurements published for isolated cardiac mitochondrial membranes. There is no evidence in the cells for contamination of CPT-I activities by extramitochondrial sources, in particular, the sarcoplasmic reticulum (SR). The presence of carnitine octanoyltransferase (COT) is not detected either in the cells or in preparations of adult SR from which COT is subsequently isolated. With these control measurements, the inhibition kinetics of CPT-I in the cardiac cells in situ maintains a partial competitive pattern which is more pronounced with decanoyl-CoA than with palmitoyl-CoA as substrate. The presence of a malonyl-CoA/long chain acyl-CoA binding site on CPT-I, distinct from the inhibitory site, has previously been proposed. Existence of this binding region is consistent with partial inhibition kinetics so that malonyl-CoA at this site could modify the CPT-high-affinity malonyl-CoA inhibitory interaction, producing acylcarnitine even at high malonyl-CoA concentrations in the cell. These findings may help to explain, in part, the inability to suppress completely beta-oxidation in the heart where malonyl-CoA may be 50 to 100 times the estimated values of its Ki.
在分离的线粒体中，丙二酰辅酶A与肉碱棕榈酰转移酶（CPT-I）相互作用的机制因膜片段化和CPT-II暴露而变得复杂。利用培养的新生大鼠心肌细胞，开发了一种原位模型来测量CPT-I。在用5 microM洋地黄皂苷处理的心肌细胞中，根据测定条件下受损心肌细胞中柠檬酸合酶的活性定量，CPT-II对CPT活性的污染为0.62%。此外，心肌细胞CPT-I对丙二酰辅酶A的敏感性、其对癸酰辅酶A的底物偏好以及CPT-I对左旋肉碱的亲和力（0.19 mM）与已发表的关于分离的心脏线粒体膜的类似测量结果相当。在细胞中没有证据表明CPT-I活性受到线粒体外来源的污染，特别是肌浆网（SR）。在细胞或随后从中分离出肉碱辛酰转移酶（COT）的成年SR制剂中均未检测到COT的存在。通过这些对照测量，原位心肌细胞中CPT-I的抑制动力学维持部分竞争模式，以癸酰辅酶A为底物时比以棕榈酰辅酶A为底物时更为明显。先前有人提出CPT-I上存在一个与抑制位点不同的丙二酰辅酶A/长链酰基辅酶A结合位点。该结合区域的存在与部分抑制动力学一致，因此该位点的丙二酰辅酶A可以改变CPT与高亲和力丙二酰辅酶A的抑制相互作用，即使在细胞中丙二酰辅酶A浓度很高时也能产生酰基肉碱。这些发现可能有助于部分解释在心脏中无法完全抑制β氧化的原因，在心脏中丙二酰辅酶A的浓度可能是其Ki估计值的50至100倍。
