Insulin receptors on isolated heart cells: effect of temperature and hydrolytic enzymes

Isolated muscle cells from adult rat heart have been used to study the effect of temperature and enzymic digestion on the binding of 125I-labelled insulin. Equilibrium binding studies were performed at both 4 and 37 degrees C, using insulin concentrations ranging from 2.5 X 10(-11) mol/l to 10(-6) mol/l. The empty site affinity constant decreased by 51% from 1.0 X 10(8) l/mol at 4 degrees C to 4.9 X 10(7) l/mol at 37 degrees C, whereas the total receptor concentration remained unaltered at both temperatures. The rate of dilution induced dissociation was enhanced by the presence of native insulin at 37 degrees C, confirming the presence of negative cooperativity among the receptor sites at physiological temperatures. Treatment of isolated heart cells with trypsin and beta-galactosidase led to a decrease in specific binding of 125I-labelled insulin. Myocytes treated with neuraminidase exhibited a significant increase in insulin binding, which was shown to be due to an increase in insulin-receptor affinity. These studies provide new information on the molecular characteristics of insulin receptors in the heart muscle.