Skip to main content
NCBI 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
. 1984 Jul;81(13):4217–4221. doi: 10.1073/pnas.81.13.4217

Solubilization and characterization of mu, delta, and kappa opioid binding sites from guinea pig brain: physical separation of kappa receptors.

Y Itzhak, J M Hiller, E J Simon
PMCID: PMC345400  PMID: 6330751

Abstract

Sucrose density gradient centrifugation of digitonin-solubilized opioid binding sites from guinea pig brain and cerebellum was carried out. Centrifugation of extracts of whole brain into a gradient devoid of sodium and low in digitonin revealed the presence of two well-separated peaks of opioid binding activity. Peak A was shown to have the binding characteristics of kappa sites, whereas peak B seems to be a mixture of mu and delta sites. When extracts of guinea pig cerebellum were treated in the same manner, a single peak of binding activity was obtained that coincided with peak A from guinea pig brain and exhibited the characteristics of kappa binding sites. All three sites closely resemble their membrane-bound counterparts, retaining good affinity and selectivity for their appropriate ligands. The apparent sedimentation coefficients (S20,w) of the digitonin-solubilized binding sites present in the two peaks are 19 s for peak A and 34-39 s for peak B, and the estimated apparent molecular weights are 400,000 for kappa sites and 750,000-875,000 for the mixture of mu and delta sites. Our results suggest that kappa sites constitute separate molecular entities from mu and delta sites.

Full text

PDF
4217

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. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brady K. T., Balster R. L., May E. L. Stereoisomers of N-allylnormetazocine: phencyclidine-like behavioral effects in squirrel monkeys and rats. Science. 1982 Jan 8;215(4529):178–180. doi: 10.1126/science.6274022. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Chow T., Zukin R. S. Solubilization and preliminary characterization of mu and kappa opiate receptor subtypes from rat brain. Mol Pharmacol. 1983 Sep;24(2):203–212. [PubMed] [Google Scholar]
  7. Clark R. W. Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients, an improved, simplified method. Biochim Biophys Acta. 1976 Apr 23;428(2):269–274. doi: 10.1016/0304-4165(76)90034-9. [DOI] [PubMed] [Google Scholar]
  8. Cuatrecasas P. Isolation of the insulin receptor of liver and fat-cell membranes (detergent-solubilized-( 125 I)insulin-polyethylene glycol precipitation-sephadex). Proc Natl Acad Sci U S A. 1972 Feb;69(2):318–322. doi: 10.1073/pnas.69.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilbert P. E., Martin W. R. The effects of morphine and nalorphine-like drugs in the nondependent, morphine-dependent and cyclazocine-dependent chronic spinal dog. J Pharmacol Exp Ther. 1976 Jul;198(1):66–82. [PubMed] [Google Scholar]
  10. HUBBARD R. The molecular weight of rhodopsin and the nature of the rhodopsin-digitonin complex. J Gen Physiol. 1954 Jan 20;37(3):381–399. doi: 10.1085/jgp.37.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hammonds R. G., Jr, Nicolas P., Li C. H. Characterization of beta-endorphin binding protein (receptor) from rat brain membranes. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6494–6496. doi: 10.1073/pnas.79.21.6494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hiller J. M., Angel L. M., Simon E. J. Multiple opiate receptors: alcohol selectively inhibits binding to delta receptors. Science. 1981 Oct 23;214(4519):468–469. doi: 10.1126/science.6270788. [DOI] [PubMed] [Google Scholar]
  13. Holtzman S. G. Phencyclidine-like discriminative effects of opioids in the rat. J Pharmacol Exp Ther. 1980 Sep;214(3):614–619. [PubMed] [Google Scholar]
  14. Howells R. D., Gioannini T. L., Hiller J. M., Simon E. J. Solubilization and characterization of active opiate binding sites from mammalian brain. J Pharmacol Exp Ther. 1982 Sep;222(3):629–634. [PubMed] [Google Scholar]
  15. Itzhak Y., Bonnet K. A., Groth J., Hiller J. M., Simon E. J. Multiple opiate binding sites in human brain regions: evidence for kappa and sigma sites. Life Sci. 1982 Sep 20;31(12-13):1363–1366. doi: 10.1016/0024-3205(82)90382-4. [DOI] [PubMed] [Google Scholar]
  16. Itzhak Y., Hiller J. M., Gioannini T. L., Simon E. J. Effect of digitonin on the binding of opiate agonists and antagonists to membrane-bound and soluble opioid binding sites. Brain Res. 1984 Jan 23;291(2):309–315. doi: 10.1016/0006-8993(84)91263-0. [DOI] [PubMed] [Google Scholar]
  17. Itzhak Y., Hiller J. M., Gioannini T. L., Simon E. J. Evidence for the solubilization of multiple opioid binding sites from mammalian brain. Life Sci. 1983;33 (Suppl 1):191–194. doi: 10.1016/0024-3205(83)90475-7. [DOI] [PubMed] [Google Scholar]
  18. Itzhak Y., Kalir A., Sarne Y. On the opioid nature of phencyclidine and its 3-hydroxy derivative. Eur J Pharmacol. 1981 Jul 17;73(2-3):229–233. doi: 10.1016/0014-2999(81)90097-2. [DOI] [PubMed] [Google Scholar]
  19. James I. F., Chavkin C., Goldstein A. Preparation of brain membranes containing a single type of opioid receptor highly selective for dynorphin. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7570–7574. doi: 10.1073/pnas.79.23.7570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kosterlitz H. W., Paterson S. J. Characterization of opioid receptors in nervous tissue. Proc R Soc Lond B Biol Sci. 1980 Oct 29;210(1178):113–122. doi: 10.1098/rspb.1980.0122. [DOI] [PubMed] [Google Scholar]
  21. Kosterlitz H. W., Paterson S. J., Robson L. E. Characterization of the kappa-subtype of the opiate receptor in the guinea-pig brain. Br J Pharmacol. 1981 Aug;73(4):939–949. doi: 10.1111/j.1476-5381.1981.tb08749.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lord J. A., Waterfield A. A., Hughes J., Kosterlitz H. W. Endogenous opioid peptides: multiple agonists and receptors. Nature. 1977 Jun 9;267(5611):495–499. doi: 10.1038/267495a0. [DOI] [PubMed] [Google Scholar]
  23. Martin W. R., Eades C. G., Thompson J. A., Huppler R. E., Gilbert P. E. The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther. 1976 Jun;197(3):517–532. [PubMed] [Google Scholar]
  24. Paterson S. J., Robson L. E., Kosterlitz H. W. Classification of opioid receptors. Br Med Bull. 1983 Jan;39(1):31–36. doi: 10.1093/oxfordjournals.bmb.a071787. [DOI] [PubMed] [Google Scholar]
  25. Quirion R., Hammer R. P., Jr, Herkenham M., Pert C. B. Phencyclidine (angel dust)/sigma "opiate" receptor: visualization by tritium-sensitive film. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5881–5885. doi: 10.1073/pnas.78.9.5881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robson L. E., Kosterlitz H. W. Specific protection of the binding sites of D-Ala2-D-Leu5-enkephalin (delta-receptors) and dihydromorphine (mu-receptors). Proc R Soc Lond B Biol Sci. 1979 Aug 31;205(1160):425–432. doi: 10.1098/rspb.1979.0076. [DOI] [PubMed] [Google Scholar]
  27. Ruegg U. T., Cuenod S., Hiller J. M., Gioannini T., Howells R. D., Simon E. J. Characterization and partial purification of solubilized active opiate receptors from toad brain. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4635–4638. doi: 10.1073/pnas.78.7.4635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rüegg U. T., Hiller J. M., Simon E. J. Solubilization of an active opiate receptor from Bufo marinus. Eur J Pharmacol. 1980 Jun 27;64(4):367–368. doi: 10.1016/0014-2999(80)90247-2. [DOI] [PubMed] [Google Scholar]
  29. Simon E. J., Bonnet K. A., Crain S. M., Groth J., Hiller J. M., Smith J. R. Recent studies on interaction between opioid peptides and their receptors. Adv Biochem Psychopharmacol. 1980;22:335–346. [PubMed] [Google Scholar]
  30. Simon E. J., Hiller J. M., Edelman I. Solubilization of a stereospecific opiate-macromolecular complex from rat brain. Science. 1975 Oct 24;190(4212):389–390. doi: 10.1126/science.1182043. [DOI] [PubMed] [Google Scholar]
  31. Simon E. J., Hiller J. M., Groth J., Edelman I. Further properties of stereospecific opiate binding sites in rat brain: on the nature of the sodium effect. J Pharmacol Exp Ther. 1975 Mar;192(3):531–537. [PubMed] [Google Scholar]
  32. Simonds W. F., Koski G., Streaty R. A., Hjelmeland L. M., Klee W. A. Solubilization of active opiate receptors. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4623–4627. doi: 10.1073/pnas.77.8.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith J. R., Simon E. J. Selective protection of stereospecific enkephalin and opiate binding against inactivation by N-ethylmaleimide: evidence for two classes of opiate receptors. Proc Natl Acad Sci U S A. 1980 Jan;77(1):281–284. doi: 10.1073/pnas.77.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zajac J. M., Gacel G., Petit F., Dodey P., Rossignol P., Roques B. P. Deltakephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr: a new highly potent and fully specific agonist for opiate delta-receptors. Biochem Biophys Res Commun. 1983 Mar 16;111(2):390–397. doi: 10.1016/0006-291x(83)90318-2. [DOI] [PubMed] [Google Scholar]
  35. Zukin R. S., Zukin S. R. Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites. Mol Pharmacol. 1981 Sep;20(2):246–254. [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