Receptor binding of insulin-like growth factor-I to mammary microsomes from non-pregnant, pregnant and lactating sheep.
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.
Winder
S J
SJ
AFRC Institute of Grassland and Environmental Research, Hurley, Maidenhead, Berkshire, U.K.
Wheatley
S D
SD
Forsyth
I A
IA
D000818:Animals; D001665:Binding Sites; D005260:Female; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007774:Lactation / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D012756:Sheep / Q000378:metabolism*
D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1