用螯合剂分离的小鼠肝细胞的胰岛素结合。

Acta physiologica latino americana Pub Date : 1981-01-01
J C Cresto, D P Udrisar, M C Camberos, J C Basabe
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引用次数: 0

摘要

用Ca2+和K+螯合剂分离小鼠肝细胞。所有实验的细胞浓度范围为2.5 × 10(5)至1.44 × 10(6)个细胞/管。研究了胰岛素受体在2℃和20℃下的结合动力学,1.67 X 10(-11) M 125i -胰岛素在2℃下180 min达到平衡;50%结合时Ka为0.736 X 10(7) M-1 sec-1。在20℃下,平衡发生在30分钟;50%结合时Ka为7.519 X 10(7) M-1 sec-1。添加16.6 μ m天然胰岛素检测非特异性结合。结合动力学研究表明,这是一个纯双分子反应,因为常数在不同时间保持不变。在结合复合物解离的研究中,胰岛素从受体释放涉及一级动力学,50%的结合胰岛素在实验期间被释放。仅在20℃下,通过稀释或添加16.6微米天然胰岛素来研究解离。两种方法都得到了相同的结果,表明解离动力学为一级反应,半衰期为101 min, Kd为2.5 X 10(-4) sec-1。在平衡状态下研究了天然胰岛素(1.67 X 10(-10)、3.33 X 10(-10)、1.67 X 10(-9)、3.33 X 10(-9)、1.67 X 10(-8)、3.33 X 10(-8)、1.67 X 10(-7)、3.33 X 10(-7) M)对1.67 X 10(-11) M - 125i -胰岛素的竞争性抑制作用。活性结合位点间存在异质性:1个高亲和力低容量群体(2 C: K = 4.64 × 10(7) L/M, Ro = 213 × 10(-11) M;20 C: K = 2.90 X 10(8) L/M, Ro = 28.5 X 10(-11) M)和一个低亲和力和高容量(2 C: K = 6.81 X 10(7) L/M, Ro: 836 X 10(-11) M;20 C: K = 2.63 X 10(6) L/M, Ro: 1080 X 10(-11) M)。结果表明,在肝细胞分离中使用螯合剂在胰岛素受体相互作用的物理化学研究中具有价值。
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Insulin binding in mouse liver cells isolated with chelating agents.

Mouse liver cells were isolated with Ca2+ and K+ chelating agents. Cell concentrations in all experiments ranged from 2.5 X 10(5) to 1.44 X 10(6) cells/tube. The kinetics of insulin-receptor binding was studied at 2 C and 20 C. Binding of 1.67 X 10(-11) M 125I-insulin reached equilibrium at 2 C at 180 min; Ka at 50% binding was 0.736 X 10(7) M-1 sec-1. At 20 C equilibrium occurred at 30 min; Ka at 50% binding was 7.519 X 10(7) M-1 sec-1. Non-specific binding was measured by adding 16.6 microM native insulin. Kinetics studies of association point to a pure bimolecular reaction since the constant remains unaltered at different times. In studies of bound complex dissociation, insulin release from the receptor involves first order kinetics, 50% of the bound insulin becoming released during the experimental period. Dissociation was studied at 20 C only, either by dilution or addition of 16.6 microM native insulin. Both methods yielded the same result, showing the dissociation kinetics to be a first order reaction with a half-life of 101 min and Kd: 2.5 X 10(-4) sec-1. Competitive inhibition of native insulin (1.67 X 10(-10), 3.33 X 10(-10), 1.67 X 10(-9), 3.33 X 10(-9), 1.67 X 10(-8), 3.33 X 10(-8), 1.67 X 10(-7), 3.33 X 10(-7) M) against 1.67 X 10(-11) M 125I-insulin was studied in equilibrium. Heterogeneity among active binding sites was found: one population of high affinity and low capacity (2 C: K = 4.64 X 10(7) L/M, Ro = 213 X 10(-11) M; 20 C: K = 2.90 X 10(8) L/M Ro = 28.5 X 10(-11) M) and one of low affinity and high capacity (2 C: K = 6.81 X 10(7) L/M Ro: 836 X 10(-11) M; 20 C: K = 2.63 X 10(6) L/M, Ro: 1080 X 10(-11) M). The results show the use of chelating agents in the separation of liver cells to be of value in physicochemical studies of insulin-receptor interaction.

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