Physical Characteristics of Insulin Receptors on Renal Cell Membranes

Abstract

Certain physical characteristics of the insulin-kidney-membrane interaction in dog renal cortex have been defined. Two classes of binding sites were identified: the high-affinity-low-capacity site had a Ka of 1.3 × 109 M1 and a capacity of 43 fmols per milligram membrane protein. The lower-affinity site Ka was 3.7×107 M-1. Saturabili t y of receptor sites could not be demonstrated at any insulin concentration. At 21° C, binding of insulin was maximal after 60 minutes of incubation, with a t1/2of <1.0 minute. Specific binding of 125I-insulin was proportional to protein concentration when the fraction of insulin bound was not excessive. Proinsulin and desoctapeptide insulin displaced labeled insulin in proportion to their known biologic activities (insulin>proinsulin>desoctapep-tide insulin). Arginine vasopressin and glucagon did not compete with native (labeled) insulin for binding sites. The kinetic properties of the kidney insulin-receptor sites are similar to those previously reported for liver and fat cell receptors. A comparison of specific binding of 125I-insulin to kidney and liver membranes in the presence of various cation concentrations showed no enhancement of binding in either tissue by NaCl as high as 2 M and KC1 up to 50 mM. In contrast, 25 mM CaCl2 caused an increase in specific binding from 3.8 to 7.7 fmols 125I-insulin per milligram protein in kidney and from 1.0 to 2.1 fmols per milligram protein in liver. MgCl2 (10 mM) increased binding of insulin from 3.5 to 7.3 fmols per milligram in kidney and from 1.0 to 2.1 fmols per milligram in liver. EDTA (2mM) had no intrinsic effect on binding but reversed (by 50 per cent) the Ca2+ -mediated enhancement of binding. Divalent cations appeared to influence both high and low affinity sites. 125I-insulin degradation occurred as a function of total insulin concentration in the kidney membrane system and reduced the amount of “bindable” free hormone. The process was not saturable below 10-6 M insulin. Degradation effects influenced calculated equilibrium constants to a minimal degree.