Separation of rabbit mammary-gland prolactin receptors by ion-exchange chromatography, h.p.l.c.-gel filtration and ultracentrifugation

Abstract

Rabbit mammary-gland prolactin (Prl) receptors in the microsomal fraction were solubilized in 7.5 mM-Chaps) or 1% Triton X-100 and analysed by ion-exchange chromatography using DEAE-Bio-Gel A. Prl receptors in the presence of 7.5 mM-Chaps were separated into two different fractions (Fr. A and B), both of which showed identical specificity of binding to peptide hormones as those in the Chaps or Triton extract. oPrl and human growth hormone (hGH) bound to the same site, but other non-lactogenic hormones (follicle-stimulating hormone, oGH, luteinizing hormone and insulin) failed to bind to the Prl receptors. The dissociation constant (Kd) for Prl binding to the receptors in Fr. A was about 50% of those in Fr. B, suggesting that the rabbit mammary gland contains two types of Prl receptors, one with a high, and one with a low, Kd for Prl binding. A decrease in the concentration of Chaps in the column buffer to 4 mM caused aggregation of the receptors in Fr. A. H.p.l.c.-gel filtration, using Shim pack 150 and 300 columns connected in series, separated the receptor as a protein with an Mr of 74,000 +/- 4,900 (mean +/- S.D.) in the presence of 5 mM-Chaps, or of 36,800 +/- 2,100 in the presence of 7.5 mM-Chaps. Sucrose-gradient-centrifugation analysis showed that the Prl-receptor complexes in the presence of 5 mM-Chaps were sedimented between gamma-globulin and bovine serum albumin (5.56 +/- 0.22 S). As the Chaps concentration was increased to 7.5 mM, a further peak of the Prl-receptor complexes (4.01 +/- 0.23 S) appeared below ovalbumin. The present data suggest that the binding subunit causes the monomeric subunit to aggregate with itself or with another specific associated protein of similar Mr.

Monoclonal antibody detection of prolactin-binding subunits in the rabbit mammary gland

“…Curvilinear Scatchard plots of monoclonal antibody (MAb) binding to PRL receptors were reported (Katoh et al, 1985a;Sakai & Murakami, 1987). We showed that PRL receptors in the rabbit mammary gland can be fractionated, by ion-exchange chromatography, into two different species both of which have high PRL binding specificity and sensitivity (Sakai et al, 1986). It is, therefore, important to characterize the structural properties of the PRL receptor for the further study of PRL action in the PRL target organ, mammary gland.…”

Two Separate Receptors for Prolactin in the Rabbit Mammary Gland.

“…In the autoradiogram of the microsome PRL receptors, the band of the 83,200 species was more strongly labeled than the two other bands. In previous experiments (Sakai et al, 1986), the low Kd type-PRL receptors were completely solubilized by 7.5 mM Chaps, the high Kd type-receptors being left in the microsome fraction. These results suggest that PRL receptors in the lactating mammary gland are mostly of the high Kd type.…”

“…We have shown that Chaps-solubilized PRL receptors can be separated by ionexchange chromatography into two species, both of which had specificity and high affinity for PRL binding (Sakai et al, 1986).…”

Thermodynamic analysis of the interaction of prolactin with its receptor in the rabbit mammary-gland microsomes

“…Ovine PRL 30 i.u./mg) was kindly supplied by NIADDK, Bethesda, MD, U.S.A. PRL was iodinated by using lactoperoxidase and H202 (Sakai et al, 1986), and the specific radioactivity of 125I-PRL was about 2.2 MBq/1sg.…”

“…Microsomes (75 ,ug of protein/tube) were incubated with 125I-PRL (30000 c.p.m.) in a total volume of 0.5 ml (Sakai et al, 1986). For the Scatchard (1949) 19,23,27, and 31 'C.…”