Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor.
When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.
Whitson
R H
RH
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Grimditch
G K
GK
Sternlicht
E
E
Kaplan
S A
SA
Barnard
R J
RJ
Itakura
K
K
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D007329:Insulin Antagonists / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D009928:Organ Specificity; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism*
D007329:Insulin Antagonists; D046911:Macromolecular Substances; D011972:Receptor, Insulin