title: Insulin binding to bovine mammary membranes: comparison of microsomes versus smooth membranes.
abstract: The study was undertaken to determine if membrane preparations of bovine mammary tissue bound insulin. If binding occurred, it was also the intent to compare binding kinetics between microsomes and smooth membranes. Insulin binding to bovine mammary membranes attained equilibrium, was saturable and was specific for insulin. Additional studies showed binding to be pH sensitive and maximal at 10 mM calcium. Binding affinity of insulin to microsomes and smooth membranes was similar, with the exception that smooth membranes bound 1.8 times more insulin per unit of membrane protein than microsomes. Two different methods were used to generate data for kinetic analysis of the insulin-receptor interaction in microsomes. Competitive binding assays (.6 ng [125I]insulin plus 0 to 100 ng insulin) indicated the presence of two binding sites with dissociation constants (Kd) of .32 and 15.8 nM. Direct titration of microsomes with [125I]insulin (.02 to 10 ng/ml) revealed two binding sites with Kd of .017 and .31 nM. The affinity of the second binding site measured by the competitive binding assay method (Kd of 15.8 nM) is low and therefore may not be of physiological importance for insulin action. Insulin appears to bind to two high-affinity receptor sites in bovine mammary microsomes with Kds of .017 nM and .32 nM. These findings show that bovine mammary tissue contains receptors for insulin. In addition, isolation of smooth membranes from microsomes enriches the number of insulin receptors per unit of membrane protein without altering their binding characteristics.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*
chemical_list: D007328:Insulin; D011972:Receptor, Insulin