Unmasking of insulin receptors in rat submaxillary gland microsomes: effect of high ionic strength, phospholipase C and S-adenosyl-L-methionine.
The specific binding of [125I]insulin to submaxillary gland microsomes was significantly enhanced by increasing the ionic strength of the incubation medium. This effect was neither related to changes in receptor or hormone degradation nor in the polymerization of the tracer. When equilibrium binding data from competition curves of unlabelled insulin versus [125I]insulin were analyzed, a marked increase in total binding capacity in high ionic strength was observed (from 890 to 2440 fmol/mg protein), with no change in binding affinity. Phospholipase C digestion was also able to increase specific [125I]insulin binding to microsomes. These results suggest the presence of masked receptors in submaxillary gland microsomes. Methylation of rat submaxillary gland microsomes by using S-adenosyl-L-methionine as the methyl donor significantly increased [125I]insulin binding. Scatchard analysis of the equilibrium binding data showed that addition of S-adenosyl-L-methionine (0.46 mM) to microsomes resulted in an enhancement of the total binding capacity (from 990 to 1520 fmol/mg protein) with no change in the affinity constants, which suggests the exposure of masked insulin receptors under such conditions. Both the methyl group incorporation into membrane phospholipids and the effect on insulin binding were dependent on the S-adenosyl-L-methionine concentration used and were partially suppressed in the presence of S-adenosyl-L-homocysteine, a specific competitive inhibitor of the methyltransferases activity. When microsomes were treated with S-adenosyl-L-[methyl-3H]methionine, the 3H-labelled methyl groups incorporated were found mainly in the lipid fraction associated to phosphatidylcholine, suggesting in this case that the unmasking of insulin receptors could be a consequence of alterations produced in membrane composition. The effects of phospholipase C, S-adenosyl-L-methionine and high ionic strength on insulin binding were not additive, suggesting that these procedures unmask receptors from the same pool.
D000818:Animals; D005260:Female; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008745:Methylation; D008861:Microsomes / Q000378:metabolism; D009994:Osmolar Concentration; D010740:Phospholipases / Q000494:pharmacology*; D051381:Rats; D011972:Receptor, Insulin / Q000187:drug effects* / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology*; D013363:Submandibular Gland / Q000378:metabolism*; D010738:Type C Phospholipases / Q000494:pharmacology*
D007328:Insulin; D012436:S-Adenosylmethionine; D011972:Receptor, Insulin; D010740:Phospholipases; D010738:Type C Phospholipases