Solubilization of functional receptors for parathyroid hormone and parathyroid hormone-related peptide from clonal rat osteosarcoma cells, ROS17/2.8.
ROS17/2.8 cells, a cell line derived from a rat osteosarcoma, have abundant receptors for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). A particulate membrane fraction was prepared from these cells and it was solubilized using relatively mild conditions with digitonin (0.25%), a nonionic detergent. When radioligands of both PTH and PTHrP were incubated with this membrane fraction in the absence of any protease inhibitor at 15 degrees C, approximately 75% of these radioligands were degraded within 2 hours. This degradative activity was inhibited more effectively by bacitracin than by any of several other protease inhibitors tested. The digitonin-solubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr36]PTHrP(1-36) amide (PTHrP(1-36)) and N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), as cross-linker. When an aliquot of the reaction solution was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, a broad band was observed that had an apparent molecular size of 90,000 daltons (M(r) = 90 kD). This band was no longer seen when the binding was conducted in the presence of 10(-6) M of unlabeled PTHrP(1-36), and it was decreased in density when binding was conducted in the presence of 10(-6) M of unlabeled [Nle8,18, Tyr34] bovine PTH(1-34) amide (NlePTH). The solubilized receptors retained their capacity to bind the radioligand after partial purification by wheat-germ agglutinin affinity-chromatography. The use of relatively mild detergent conditions thus offers a means to solubilize receptors that retain their capacity to bind PTH and PTHrP.
Uneno
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Department of Medicine, Massachusetts General Hospital, Boston.
Yamamuro
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Jüppner
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Abou-Samra
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Keutmann
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Potts
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JT
Segre
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D000818:Animals; D001345:Autoradiography; D001414:Bacitracin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D012516:Osteosarcoma / Q000737:chemistry*; D010281:Parathyroid Hormone / Q000378:metabolism*; D044162:Parathyroid Hormone-Related Protein; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D018016:Receptors, Parathyroid Hormone; D012995:Solubility; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011480:Protease Inhibitors; D011506:Proteins; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D001414:Bacitracin