Solubilization of prolactin receptor by a Zwitterionic detergent

Abstract

A prolactin receptor was purified from a solu-bilized preparation of mouse microsomal membranes by affinity chromatography. The receptors were solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfo-nate) (Chaps) a zwitterionic derivative of cholic acid. The affinity gel was prepared by coupling ovine prolactin to CH-Sepharose 4B. After extensive elution of the nonspecifically adsorbed proteins, the receptors were eluted with 4 M urea/i M NaCI/0.5% Chaps followed by 5 M MgCl2/0.5% Chaps. The eluted receptor appeared on NaDodSO4/polyacrylamide gels as a single major band of Mr 37,000. The purified receptor retained its specificity for lactogenic hormones and binds "25I-labeled ovine prolactin with aKa of 2-6 x 109 M-1. Prolactin (PRL) plays a key role in development and differentiation of normal mammary glands (1) and in mammary tumor-igenesis (2). The initial step in PRL action is the interaction of this hormone with its receptor in the membrane. In the past, most studies attempting to understand the mechanism of PRL action have required that the receptor be examined solely by its ability to bind to lactogenic hormone. From these binding data, the mode of regulation of the receptor itself has been inferred. However, these observations have been based on the functionality of the receptor molecule. To effectively probe the nature of regulation of the PRL receptor itself, it is most desirable to have specific antibodies directed against it. To this end, we undertook the purification of a PRL receptor, using mouse liver as the target tissue and membrane source. This tissue contains both somatogenic and lactogenic hormone receptors (3). Previous attempts at purification of the lactogenic hormone (or PRL) receptor from target tissues have utilized Triton X-100 as the solubilization agent (4, 5). Because PRL aggregates in the presence of this detergent (4), human growth hormone (hGH) had to be used as the binding ligand. As a result, these studies have been hampered by the ability of hGH to bind to both so-matogenic and lactogenic hormone receptors (4, 6-8). Recently (9), we succeeded in solubilizing an active PRL receptor from mouse liver microsomal membranes by using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-pro-panesulfonate (Chaps), a zwitterionic derivative of cholic acid (10). The solubilized material had the same binding affinity and specificity as those of the membrane receptor (9). Because PRL does not aggregate in the presence of Chaps and, thus, can effectively be utilized as a binding ligand (9), we attempted the purification ofthe PRL receptor, using an affinity column prepared by conjugating ovine PRL (oPRL) to Sepha-rose beads. The purified material, which consists of a major Mr 37,000 peptide, retains its specificity for lactogenic hormones and binds oPRL with the same high affinity as the membrane-bound receptor. MATERIALS AND METHODS Materials. Chemicals were from the following sources: oPRL (NIH-P-S-14) and bovine growth hormone (bGH; NIH-GH-B-18) were from t...

Surfactant-Mediated Solubilization of Myelin Figures: A Multistep Morphological Cascade

“…In our study, the solubilizing detergent of choice was the synthetic zwitterionic surfactant Zwittergent 3–12 ( n -dodecyl- N , N -dimethyl-3-ammonio-1-propanesulfonate or DDAPS, CMC = 2–4 mM), which is used widely in the isolation of membrane proteins. − Both single-lipid component and multicomponent myelins were studied. Single-component myelins were prepared using fluid-phase 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), and multicomponent myelins comprised an equimolar mixture of POPC, cholesterol (CHOL), and egg sphingomyelin (SM).…”