Developmental changes in insulin receptors of pig red blood cells

Scatchard analysis of the insulin binding to pig reticulocytes, fetal red cells, and adult erythrocytes showed the maximum number of high-affinity binding sites per cell to be 274, 147, and 29, respectively. All three cell types displayed a practically identical dissociation constant of approximately 1.22 X 10(-8) M at the high-affinity region. A long-term in vitro incubation of the fetal red cells and reticulocytes under tissue culture conditions was accompanied by a significant loss of insulin-binding capacity without any appreciable alteration of the dissociation constant. The isolation and characterization of insulin-receptor complexes from these cell types were carried out to establish whether the difference in insulin-binding capacity was due to the difference in the amount of the same species or due to different species of insulin receptors. Membrane proteins were extracted with Triton X-102 and fractionated by DEAE-Sephacel ion-exchange column chromatography. Each peak sample was complexed with 125I-insulin, and the complexes were covalently crosslinked and then applied to a Sepharose CL-6B column. A 95,000-Da complex was obtained from adult pig erythrocyte membranes; 220,000- and and 95,000-Da complex was obtained from adult pig erythrocyte membranes; 220,000- and 95,000-Da complexes from pig reticulocyte membranes; and greater than 600,000-, 220,000-, and 95,000-Da complexes from pig fetal cell membranes. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under a nonreducing condition, the 95,000-Da complex was dissociated into a 53,000-Da component; the greater than 600,000-Da complex into greater than 320,000-, 130,000-, and 53,000-Da components; and the 220,000-Da complex was dissociated into 220,000-, 130,000-, and 53,000-Da components. These findings strongly suggest that the decrease in insulin binding during the developmental changes of red blood cells is due to a disappearance of high-molecular-weight insulin receptors rather than a decrease in the amount of the smaller receptor molecules.