BINDING OF MALONYL-COA TO ISOLATED-MITOCHONDRIA - EVIDENCE FOR HIGH-AFFINITY AND LOW-AFFINITY SITES IN LIVER AND HEART AND RELATIONSHIP TO INHIBITION OF CARNITINE PALMITOYLTRANSFERASE ACTIVITY
BINDING OF MALONYL-COA TO ISOLATED-MITOCHONDRIA - EVIDENCE FOR HIGH-AFFINITY AND LOW-AFFINITY SITES IN LIVER AND HEART AND RELATIONSHIP TO INHIBITION OF CARNITINE PALMITOYLTRANSFERASE ACTIVITY
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DOI：
10.1042/bj2220639
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发表时间：
1984-01-01
影响因子：
4.1
通讯作者：
SAGGERSON, ED
中科院分区：
文献类型：
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作者：
BIRD, MI;SAGGERSON, ED
[14C]丙二酰辅酶A与从大鼠肝脏和心脏分离的完整线粒体结合，其方式与每个组织中存在2类独立的结合位点一致。从喂养的雄性大鼠获得的线粒体的结合特征是：对于心脏，KD(1)=11-18nM，KD(2)=30μM，N1[最大结合能力]=7pmol/mg蛋白质，N2=.apprx。 660 pmol/mg 蛋白质；对于肝脏，KD(1) = 0.1 μM，KD(2) = 5.6 μM，N1 = 11 pmol/mg 蛋白质，N2 = 165 pmol/mg 蛋白质。在存在 40 μM-棕榈酰-CoA 的情况下，高亲和力位点处的结合特征发生改变，因此对于心脏 KD(1) = 0.26 μM，N1 没有变化，对于肝脏 KD(1) = .apprx。 2 .mu.M，N1 增加至 .apprx。 40 pmol/mg 蛋白质。两种组织在高亲和力位点丙二酰辅酶 A 结合紧密度方面的差异解释了心脏 CPT1（肉毒碱棕榈酰转移酶的明显形式）对丙二酰辅酶 A 抑制的显着更高的敏感性。饥饿（24小时）不会改变[14C]丙二酰辅酶A与肝线粒体结合的特征，也不会改变棕榈酰辅酶A置换[14C]丙二酰辅酶A的I50（产生50%抑制的浓度）。因此，饥饿时肝脏 CPT1 对丙二酰辅酶 A 抑制的敏感性降低并不能用丙二酰辅酶 A 结合的差异来解释。丙二酰辅酶 A 对心脏线粒体高亲和力位点的占据百分比与在类似条件下测量的 CPT1 抑制百分比密切相关。这一发现支持了以下观点：高亲和力结合位点是介导丙二酰辅酶A抑制CPT1的功能位点。肝线粒体的类似实验也表明，丙二酰辅酶 A 占据高亲和力位点可调节 CPT1 活性。 5,5''-二硫双-(2-硝基苯甲酸)可降低肝心CPT1对丙二酰辅酶A抑制的敏感性，也降低[14C]丙二酰辅酶A与心脏线粒体高亲和力位点的结合。 [14C]丙二酰辅酶A与肝线粒体高亲和力位点结合的N1值在各种生理状态下测定，其中包括CPT1最大活性的7倍范围（进食、饥饿、怀孕、甲状腺功能减退、胎儿）。 N1 值在这些状态下没有变化。这一发现支持了高亲和力位点不太可能是 CPT1 催化单元的观点。丙二酰辅酶 A 的低亲和力结合位点的生理重要性尚不清楚。
[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of 2 independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18 nM, KD(2) = 30 .mu.M, N1 [maximal binding capacity] = 7 pmol/mg of protein, N2 = .apprx. 660 pmol/mg of protein; for liver, KD(1) = 0.1 .mu.M, KD(2) = 5.6 .mu.M, N1 = 11 pmol/mg of protein, N2 = 165 pmol/mg of protein. In the presence of 40 .mu.M-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 .mu.M, with no change in N1 and for liver KD(1) = .apprx. 2 .mu.M, with N1 increased to .apprx. 40 pmol/mg of protein. Differences between the 2 tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA. Starvation (24 h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5''-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart of liver CPT1 to inhibition by malonyl-CoA, also decreased [14C]malonyl-CoA binding to high affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, fetal). The N1 value did not change in these states. This finding supports the proposal that the high-affinity site is unlikely to be the catalytic unit of CPT1. The physiological importance of the low-affinity binding sites for malonyl-CoA is unknown.