Immunoprecipitation of the parathyroid hormone receptor
An /sup 125/I-labeled synthetic analog of bovine parathyroid hormone, (8-norleucine,18-norleucine,34-tyrosine)PTH-(1-34) amide ((Nle)PTH-(1-34)-NH/sub 2/), purified by high-pressure liquid chromatography (HPLC), was employed to label the parathyroid hormone (PTH) receptor in cell lines derived from PTH target tissues: the ROS 17/2.8 rat osteosarcoma of bone and the CV1 and COS monkey kidney lines. After incubation of the radioligand with intact cultured cells, the hormone was covalently attached to receptors by using either a photoaffinity technique or chemical (affinity) crosslinking. In each case, covalent labeling was specific, as evidenced by a reduction of labeling when excess competing nonradioactive ligand was present. After covalent attachment of radioligand, membranes were prepared form the cells and solubilized in the nonionic detergent Nonidet P-40 or octyl glucoside. Analysis of the immunoprecipitate on NaDod-SO/sub 4//polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of a doublet of apparent molecular mass 69-70 kDa. Specifically labeled bands of approximate molecular mass 95 and 28 kDa were also observed. The anti-PTH IgG was affinity purified by passage over a PTH-Sepharose column and used to made an immunoaffinity column. These studies suggest that the use of an anti-PTH antiserum that binds receptor-bound hormone is likely to be a useful step in the further physicochemical characterization and purification of the PTH receptor.
- Research Organization:
- Merck Sharp and Dohme Research Labs., West Point, PA
- OSTI ID:
- 5723737
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:1; ISSN PNASA
- Country of Publication:
- United States
- Language:
- English
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62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMAL CELLS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
GLOBULINS
HORMONES
IMMUNOGLOBULINS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PARATHORMONE
PEPTIDE HORMONES
PROTEINS
PURIFICATION
RADIOISOTOPES
RADIORECEPTOR ASSAY
RECEPTORS
SEPARATION PROCESSES
TRACER TECHNIQUES
TUMOR CELLS