IDENTIFICATION AND CHARACTERIZATION OF IN SULIN AND EPIDERMAL GROWTH-FACTOR RECEPTORS IN LOACH EMBRYONIC-CELLS

I-125!Insulin and I-125!EGF found to be specifically and reversibly bound by loach embryonic cells isolated at the early gastrula stage (1 0-12 h of development at 21-degrees-C). The equilibrium analysis showe d that specific binding of radioligands by cells reached saturation at concentrations of insulin and EGF higher than 40 and 12 nM, respectiv ely. A Scatchard plot of insulin binding was of curvilinear character. Kd for two classes of insulin-binding sites were 7.3 10(-11) and 9. 9 10(-9) M. and their number was 115 +/- 12 and 4640 +/- 370 per cel l, respectively. EGF receptors are represented by one class of binding sites with K(d) 0.72 10(-9) M and its number was 5460 +/- 105 per c ell. After affinity labeling of plasma membrane proteins from loach em bryonic cells by iodinated ligands using bifunctional cross-linking ag ent - disuccinimidylsubetate, insulin was shown to be specifically bou nd with 125 and 115 kD proteins and EGF - with 180 kD protein. For par tial purification of insulin and EGF receptors we used affinity chroma tography on wheat germ agglutinin-sepharose. It was shown that insulin stimulated phosphorylation of 97 kD protein and EGF - 180 kD protein, which correspond to autophosphorylated forms of insulin receptor beta -subunit and EGF receptor. Our results prove that the early loach embr yonic cells possess the functional receptors of insulin and EGF. The p ossible role of above mentioned polypeptide growth factors in the regu lation of cell proliferation and differentiation in early embryogenesi s is discussed.