Abstract
Insulin receptors were solubilized from rat liver microsomes by the
nonionic detergent Triton X-100. After gel filtration of the extract
on Sepharose CL-6B, two insulin-binding species (peak I and peak
II) were obtained. The structure and binding properties of both peaks
were characterized. Gel filtration yielded Stokes radii of 9.2 nm
(peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At
4 degrees C peak I showed optimal insulin binding at pH 8.0 and high
ionic strength. In contrast, peak II had its binding optimum at pH
7.0 and low ionic strength, where peak I binding was minimal. For
peak I the change in insulin binding under different conditions of
pH and ionic strength was due to a change in receptor affinity only.
For peak II an additional change in receptor number was found. Both
peaks yielded non-linear Scatchard plots under most of the buffer
conditions examined. At their binding optima at 4 degrees C the high
affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM
(peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis
of peak I revealed five receptor bands with Mr 400 000, 365 000,
320 000, 290 000, and 245 000 under non-reducing conditions. For
peak II two major receptor bands with Mr 210 000 and 115 000 were
found. The peak II receptor bands were also obtained after mild reduction
of peak I. After complete reduction both peaks showed one major receptor
band with Mr 130 000. The reductive generation of the peak II receptor
together with molecular mass estimations suggest that the peak I
receptor is the disulfide-linked dimer of the peak II receptor. Thus,
Triton extracts from rat liver microsomes contain two receptor species,
which are related, but differ considerably in their size and insulin-binding
properties.
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