Identification of insulin binding sites in isolated cells from rat submaxillary gland.

Isolated cells from rat submaxillary gland bound 125I-labelled insulin in a time-dependent process that reached a maximum at 30-40 min at 25 degrees C. The radioactivity bound to cells could be dissociated by dilution of the binding site-hormone complex with the incubation buffer. The presence of unlabelled insulin in the incubation buffer inhibited 125I-labelled insulin degradation according to the amount of hormone added. After 10 min of incubation at at 25 degrees C, radioactivity associated to cells was almost exclusively identified as intact 125I-labelled insulin. With increasing times, a greater contribution of final products of degradation in total radioactivity bound to cells was observed; nevertheless, in the presence of unlabelled insulin the radioactivity associated to low molecular weight products markedly decreased. Equilibrium binding data analysis gave rise to a non-linear Scatchard plot, whose high affinity component showed a dissociation constant of 6.6 +/- 0.4 nM. These observations are consistent with the presence of insulin binding sites in rat submaxillary gland cells which are similar in their characteristics to those identified in other tissues.

Effect of proteolytic enzymes on masked insulin receptors in rat submaxillary gland microsomes.

Trypsin and alpha-chymotrypsin effects on masked insulin receptors were studied. Phospholipase C treatment, incubation in a high ionic strength buffer or solubilization were used as alternative procedures for the unmasking of insulin receptors. These three methods expose receptor structures which are inaccessible to insulin in the current experimental conditions of binding assays without any significant change in binding affinity. Both exposed and masked receptors proved to be equally sensitive to trypsin and alpha-chymotrypsin degradation. At 25 degrees C, about 5 micrograms trypsin/ml for 50 min or 80 micrograms alpha-chymotrypsin/ml for 200 min were necessary in each case to cause a 50% inhibition of the binding of 125I-iodo insulin to microsomes. The results suggest that masked receptors are only nonfunctional to bind insulin but they are not located in compartments inaccessible to molecules present in the medium.

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.