肝細胞膜インスリンレセプターに関する基礎的研究

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The interaction of insulin and insulin receptor was studied with rat liver plasma membranes. Rat liver was homogenized with a Polytron PT 20 ST and fractionated by differential centrifugation. Prepared plasma membranes (100 μg protein) were incubated with enzymatically iodinated 125I-insulin (0.3 ng, specific activity 107 μCi/μg) in 25 mM Tris-HCl buffer, pH 7.5, containing 0.9% NaCl and 1% bovine serum albumin. The membranes which bound with insulin were separated from free insulin by filtration on millipore filters (EGWP) and the filters were washed with 5 ml of the ice-cold buffer. Radioactivity on the filters was counted in an autogamma spectrometer. The results obtained were as follows : 1) The 12,000xg- and 17,000xg-sediments obtained after subfractionation of liver homogenates showed almost equally high specific binding activity with 125I-insulin and less activity was detected in the 600 g-, 5,000 g- and 40,000 g- sediments and the 40,000 g- supernatant. 2) Specific binding of insulin with the membrane fraction was time-, temperature-and ionic strength-dependent. The highest binding was obtained under conditions in which the membrane fraction was incubated with insulin for 24 hours at 4°C in the buffer containing 1 M NaCl. Under these conditions, specific binding of 125I-insulin was 26.8% of the total radioactivity. 3) The effect of native insulin on the binding of 125I-insulin with the membrane fraction was studied in the range of 0-6.4 * 105 μU/ml of unlabeled insulin and a distinct competitive displacement of 125I-insulin with native insluin was observed between 10 and 104 μU/ml. 4) Kinetic studies by the Scatchard plot analysis of the above results revealed heterogeneity in insulin receptors or receptor sites, one with a high affinity of 109 M-1 order and the other with a low affinity of 108 M-1 order. Both affinities were also affected by temperature and ionic strength.
日本内分泌学会の論文

肝細胞膜インスリンレセプターに関する基礎的研究

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