Prolactin-Binding Components in Rabbit Mammary Gland: Characterization by Partial Purification and Affinity Labeling*

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Abstract
The molecular characteristics of the PRL [prolactin] receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: affinity purification of PRL receptors and direct electorphoretic analysis, and affinity cross-linking of microsomal receptors with [125I]ovine PRL ([125I]oPRL). PRL receptors were solubilized from mammary microsomes with 3-[(3-cholamidopropyl)dimethylammonio]1-propane sulfonate, and they were purified using an oPRL agarose affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS PAGE] and Ag staining of the gel revealed at least 9 bands, including a 32,000 MW band which was most intensively labeled with 125I using the chloramine-T method. Covalent labeling of PRL receptors with [125I]oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 MW was produced by all 3 cross-linkers when SDS-PAGE was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the MW of [125I]oPRL, which was estimated from the migration distance on the gel, MW of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only 1 major hormone-receptor complex band was observed. The same MW binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher MW binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed 5 [125I]oPRL-binding components, 3 of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit. From these observations, it is concluded that this predominant 32,000 MW component is a major binding subunit of the PRL receptor molecule, and it does not aggregate with itself or with other subunits through S-S linkages.