Purification of the insulin receptor protein from porcine liver membranes.

Insulin receptor protein was isolated from porcine liver membranes. Starting with 600 g of liver, the plasma membrane-enriched fraction with extracted with detergent. After AcA 34 gel chromatography, affinity chromatography and DEAE-Sephacel chromatography an insulin receptor preparation was obtained which was 2500-fold purified over crude homogenate and which had a specific binding activity of up to 1 x 10(-9) mol insulin/mg protein. The yield was 200 micrograms. Analysis of binding data according to Scatchard resulted in curvilinear plots and substantially the same affinity constants with the membrane preparation and also with the purified insulin receptor protein. Dodecyl sulfate gradient gel electrophoresis of the purified insulin receptor protein showed up to eight protein bands in the range from 38 to 175 kDa. The main bands indicate an apparent molecular mass of 38, 60, 80, 120 and 150 kDa for the insulin receptor-binding protein subunits.

Binding and degradation of insulin by plasma membranes from bovine liver isolated by a large scale preparation.

With the large-scale preparation described, as much as 1 kg of bovine liver can be processed, giving a yield of more than 1 g plasma membrane protein. From analytical and morphological criteria the plasma membrane fraction isolated mainly derives from bile-canalicular and contiguous areas of the hepatocytes. The insulin binding activity is quite similar to insulin receptors in other cell systems and membrane preparations. Insulin-degrading activity is very low in the isolated plasma fraction. Most of degrading activity is located in a microsomal membrane fraction. Nevertheless the Km and the pH dependence of the insulin-degrading activity in both fractions are nearly identical. From these studies we conclude that binding and degradation of insulin are two independent processes located on different cell organelles.