Fraser F, Corstorphine C G, Price N T, Zammit V A
Hannah Research Institute, Ayr, UK.
FEBS Lett. 1999 Mar 5;446(1):69-74. doi: 10.1016/s0014-5793(99)00179-9.
Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activity that is inhibitable by malonyl-CoA. The overt carnitine palmitoyltransferase activity (CPTo) associated with the different fractions was measured. Mitochondria accounted for 65% of total cellular CPTo activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. In parallel experiments, rat livers were perfused in situ with medium containing dinitrophenyl (DNP)-etomoxir in order to inhibit quantitatively and label covalently (with DNP-etomoxiryl-CoA) the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. In all three membrane fractions, a single protein with an identical molecular mass of approximately 88,000 kDa (p88) was labelled after DNP-etomoxir perfusion of the liver. The abundance of labelled p88 was quantitatively related to the respective specific activities of CPTo in each fraction. On Western blots the same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes of the cytosolic N- and C-domains and of the inter-membrane space loop (L) domain of the mitochondrial enzyme (L-CPT I). However, the reaction of the microsomal protein with the anti-N peptide antibody (raised against epitope Val-14-Lys-29 of CPT I) was an order of magnitude stronger than expected from either microsomal CPTo activity or its DNP-etomoxiryl-CoA labelling. This suggests that the N-terminal domain of the microsomal protein differs from that in the mitochondrial or peroxisomal protein. This conclusion was confirmed using antibody back-titration experiments, in which the binding of anti-N and anti-C antibodies by mitochondria and microsomes was quantified.
线粒体、微粒体和过氧化物酶体均表达明显的（面向胞质溶胶的）肉碱棕榈酰转移酶活性，该活性可被丙二酰辅酶A抑制。测定了与不同组分相关的明显肉碱棕榈酰转移酶活性（CPTo）。线粒体占细胞总CPTo活性的65%，微粒体和过氧化物酶体的贡献分别占其余的25%和10%。在平行实验中，用含有二硝基苯基（DNP）-依托莫昔的培养基原位灌注大鼠肝脏，以便在接近生理条件下定量抑制并共价标记（用DNP-依托莫昔酰辅酶A）每个膜系统中负责CPTo活性的分子种类。在肝脏进行DNP-依托莫昔灌注后，在所有三个膜组分中，均标记了一种分子量约为88,000 kDa的单一蛋白质（p88）。标记的p88丰度与每个组分中CPTo的各自比活性定量相关。在蛋白质免疫印迹法中，同一蛋白质与三种抗肽抗体发生免疫反应，这些抗体是针对线粒体酶（L-CPT I）的胞质溶胶N端和C端结构域以及膜间隙环（L）结构域的线性表位产生的。然而，微粒体蛋白与抗N肽抗体（针对CPT I的表位Val-14-Lys-29产生）的反应比根据微粒体CPTo活性或其DNP-依托莫昔酰辅酶A标记预期的要强一个数量级。这表明微粒体蛋白的N端结构域与线粒体或过氧化物酶体蛋白的不同。使用抗体反滴定实验证实了这一结论。在该实验中，对线粒体和微粒体与抗N和抗C抗体的结合进行了定量。
