Receptor binding of insulin-like growth factor-I to mammary microsomes from non-pregnant, pregnant and lactating sheep

Abstract

Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of 125I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0.55 nmol/l), less effectively by IGF-II (ED50 8.8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135,000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor alpha-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110-120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (Kd) 2.73 +/- 0.31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0.02) higher (Kd 0.77 +/- 0.06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 +/- 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570 +/- 52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.

Regulation of type 1 insulin-like growth factor (IGF) receptors and IGF-I mRNA by age and nutrition in ovine skeletal muscles

“…5). This is similar to the reported size for 1-5I-IGF-I complexed to the -subunit of the type 1 receptor (Massague & Czech 1982, Winder et al 1993. These data support the view that the 125I-IGF-I binding demon¬ strated by histological autoradiography is to the type 1 receptor and that it is the type 1 receptor in connective tissue which is nutritionally regulated, rather than a membrane-associated IGF-binding protein.…”

A panel of monoclonal antibodies to ovine placental lactogen

“…Microsomes were prepared by homogenization and differential centrifugation (Winder et al 1993) from the mammary glands of bromocriptine-treated New Zealand White rabbits in early lactation and the livers of Poll-Dorset ewes and were freeze dried (Parke & Forsyth 1975). Displacement studies using unlabelled ovine PL, ovine prolactin (NIH-P-S-12, 35 i.u./mg) and bovine GH (NIH-B-GH-2, 1·5 i.u./mg) confirmed that rabbit mammary gland contains predominantly lactogenic receptors (displaced by prolactin and ovine PL, but not by bovine GH, Kelly et al 1976), while sheep liver contains mainly somatotrophic receptors (displaced by bovine GH and ovine PL, but not by prolactin, Brier et al 1994).…”