Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Aug;82(15):4974–4978. doi: 10.1073/pnas.82.15.4974

Purification of the opiate receptor of NG108-15 neuroblastoma-glioma hybrid cells.

W F Simonds, T R Burke Jr, K C Rice, A E Jacobson, W A Klee
PMCID: PMC390480  PMID: 2991902

Abstract

Opiate receptors from NG108-15 neuroblastoma-glioma hybrid cell membranes were purified to apparent homogeneity in a form covalently labeled with the opiate affinity ligand 3-methylfentanylisothiocyanate (super-FIT). The purification procedure consists of five steps: quantitative labeling of opiate receptors in membranes with super-FIT; solubilization in a lubrol/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate mixture; adsorption to and elution from a wheat germ agglutinin-Sepharose column; immunoaffinity chromatography on columns of immobilized anti-fentanyl; preparative gel electrophoresis in the presence of NaDodSO4. The protein was purified approximately 30,000-fold in 3% overall yield to a state in which there is 1 mol of super-FIT bound per mol of protein, corresponding to 21,000 pmol per mg of protein, the theoretically expected specific activity. The protein is glycosylated and migrates on NaDodSO4 gel electrophoresis with a Mr near 58,000. It has a strong tendency to dimerize, even in the presence of denaturing detergents, and it exists primarily as an oligomer in nondenaturing detergents.

Full text

PDF
4978

Images in this article

4977Image
on p.4977

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bidlack J. M., Abood L. G., Osei-Gyimah P., Archer S. Purification of the opiate receptor from rat brain. Proc Natl Acad Sci U S A. 1981 Jan;78(1):636–639. doi: 10.1073/pnas.78.1.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown W. E., Howard G. C. Amino acid composition of proteins eluted from polyacrylamide gels. Methods Enzymol. 1983;91:36–41. doi: 10.1016/s0076-6879(83)91008-x. [DOI] [PubMed] [Google Scholar]
  3. Chang K. J., Cuatrecasas P. Multiple opiate receptors. Enkephalins and morphine bind to receptors of different specificity. J Biol Chem. 1979 Apr 25;254(8):2610–2618. [PubMed] [Google Scholar]
  4. Cho T. M., Ge B. L., Yamato C., Smith A. P., Loh H. H. Isolation of opiate binding components by affinity chromatography and reconstitution of binding activities. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5176–5180. doi: 10.1073/pnas.80.17.5176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gioannini T. L., Howard A., Hiller J. M., Simon E. J. Affinity chromatography of solubilized opioid binding sites using CH-Sepharose modified with a new naltrexone derivative. Biochem Biophys Res Commun. 1984 Mar 15;119(2):624–629. doi: 10.1016/s0006-291x(84)80295-8. [DOI] [PubMed] [Google Scholar]
  6. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  7. Klee W. A., Nirenberg M. A neuroblastoma times glioma hybrid cell line with morphine receptors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3474–3477. doi: 10.1073/pnas.71.9.3474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Klee W. A., Simonds W. F., Sweat F. W., Burke T. R., Jr, Jacobson A. E., Rice K. C. Identification of a Mr 58 000 glycoprotein subunit of the opiate receptor. FEBS Lett. 1982 Dec 13;150(1):125–128. doi: 10.1016/0014-5793(82)81318-5. [DOI] [PubMed] [Google Scholar]
  9. Koski G., Klee W. A. Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4185–4189. doi: 10.1073/pnas.78.7.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Law P. Y., Wu J., Koehler J. E., Loh H. H. Demonstration and characterization of opiate inhibition of the striatal adenylate cyclase. J Neurochem. 1981 May;36(5):1834–1846. doi: 10.1111/j.1471-4159.1981.tb00438.x. [DOI] [PubMed] [Google Scholar]
  13. Maneckjee R., Zukin R. S., Archer S., Michael J., Osei-Gyimah P. Purification and characterization of the mu opiate receptor from rat brain using affinity chromatography. Proc Natl Acad Sci U S A. 1985 Jan;82(2):594–598. doi: 10.1073/pnas.82.2.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  15. Milligan G., Klee W. A. The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase. J Biol Chem. 1985 Feb 25;260(4):2057–2063. [PubMed] [Google Scholar]
  16. Newman E. L., Barnard E. A. Identification of an opioid receptor subunit carrying the mu binding site. Biochemistry. 1984 Nov 6;23(23):5385–5389. doi: 10.1021/bi00318a001. [DOI] [PubMed] [Google Scholar]
  17. Nishio K., Kawakami M. Protein assays in very dilute solutions. Anal Biochem. 1982 Nov 1;126(2):239–241. doi: 10.1016/0003-2697(82)90509-7. [DOI] [PubMed] [Google Scholar]
  18. Peterson G. L. Determination of total protein. Methods Enzymol. 1983;91:95–119. doi: 10.1016/s0076-6879(83)91014-5. [DOI] [PubMed] [Google Scholar]
  19. Pfeiffer A., Herz A. Demonstration and distribution of an opiate binding site in rat brain with high affinity for ethylketocyclazocine and SKF 10,047. Biochem Biophys Res Commun. 1981 Jul 16;101(1):38–44. doi: 10.1016/s0006-291x(81)80007-1. [DOI] [PubMed] [Google Scholar]
  20. Sharma S. K., Nirenberg M., Klee W. A. Morphine receptors as regulators of adenylate cyclase activity. Proc Natl Acad Sci U S A. 1975 Feb;72(2):590–594. doi: 10.1073/pnas.72.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sutcliffe J. G., Shinnick T. M., Green N., Lerner R. A. Antibodies that react with predetermined sites on proteins. Science. 1983 Feb 11;219(4585):660–666. doi: 10.1126/science.6186024. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES