Bindung von Insulinanaloga an partiell gereinigten Insulinrezeptor aus Rattenlebermembran
- 1 January 1976
- journal article
- research article
- Published by Walter de Gruyter GmbH in Hoppe-Seyler´s Zeitschrift Für Physiologische Chemie
- Vol. 357 (1), 683-694
- https://doi.org/10.1515/bchm2.1976.357.1.683
Abstract
The insulin binding fraction was solubilized from rat liver membrane vesicles by triton and partially purified up to the specific binding activity of 2.1 pmol/mg. A further characteristic of the partially purified soluble receptor is a decrease in irreversible binding to 0.36% (.+-.0.28) with regard to total iodine insulin and to 2.3% (.+-.1.8) in comparison to reversible binding. From Scatchard plots a high affinity binding site (KD = 5 .times. 10-10 M) with low capacity (5 pmol/mg) and low affinity binding site (KD = 3 .times. 10-8 M) with high capacity (30 pmol/mg) can be seen. With carefully prepared liver membrane vesicles, the dissociation constant of the high affinity binding site from the Scatchard plot is only 2 .times. 10-9 M. With liver membrane vesicles, isolated for preparative purification procedure, the high affinity binding site could not be demonstrated. Displacement studies with insulin analogs were performed with [A1-D-alanine]insulin, [A1-L-alanine]insulin, [des-Gly-A1,NB1,NB29-(Msc)2] insulin, proinsulin and [desoctapeptidB23-B30] insulin. Results of binding measurements are presented in 1/2-maximal iodo-insulin binding, in determination of inhibitor- and dissociation constants from Dixon-, Scatchard- and Lineweaver-Burk plots. There are equal relative binding potencies of analogs, observed with crude membrane vesicles and partially purified soluble receptor, although there is a 5-fold difference in specific binding activity. Biologically active insulins are characterized by strong binding to the high affinity binding site. The binding to the low affinity binding site is not correlated to the biological activity of the insulin analog. With insulin and biologically responsive analogs a non-linear curve in the double-reciprocal Lineweaver-Burk plot can be observed. Analogs with low biological activity show a linear dependency. Functional interactions of insulin with the receptor can be demonstrated in a high affinity binding with the 1st binding site of the Scatchard plot and in a non-linear hyperbolic Lineweaver-Burk plot.This publication has 21 references indexed in Scilit:
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