Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane
Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane
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DOI：
10.1042/bj3230711
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发表时间：
1997-05-01
影响因子：
4.1
通讯作者：
Zammit, VA
中科院分区：
文献类型：
--
作者：
Fraser, F;Corstorphine, CG;Zammit, VA
用多种方法研究了大鼠肝线粒体外膜肉碱棕榈酰转移酶I(CPT I)的拓扑结构。1.通过将辛酰辅酶A和丙二酰辅酶A固定在琼脂糖珠上，使酶的活性部位和丙二酰辅酶A结合部位不能通过外膜，研究了酶的活性部位和丙二酰辅酶A结合部位从膜的胞浆侧可及的情况。两种固定化配体均能有效地与CPT I相互作用。2.采用完整或外膜因低渗肿胀(OMRM)破裂的线粒体标本，研究了蛋白酶K和胰酶对CPT I活性和丙二酰辅酶A敏感性的影响。无论是胞浆还是细胞膜两侧暴露，蛋白酶K对CPT I的活性都有显着但相似的影响。然而，在OMRM中，它对敏感性的影响更快。相比之下，胰酶仅在与OMRM孵育时降低CPT I活性。胰酶不影响残存CPT I活性的敏感性。3.用三种抗CPT I线性表位的抗肽抗体对这些处理产生的蛋白水解物进行了Western blotting分析，结果表明，在N端附近有一个蛋白水解酶K敏感部位，可从细胞膜的胞液侧进入。在完整的线粒体中没有胰酶敏感部位。在OMRM中，蛋白酶K和胰酶都是从膜间隙侧起作用的。4.用完整的线粒体和OMRM与三种抗肽抗体结合的能力来研究相应的表位在膜的胞液和膜间隙侧的可及性。5.所有这些方法的结果表明，CPT I在线粒体外膜内采用双位拓扑结构，它具有两个跨膜结构域，N-末端和C-末端都暴露在膜的胞液侧，而跨膜结构域之间的连接区突出到膜间隙。
The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.