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. 1979 Jan;76(1):204–208. doi: 10.1073/pnas.76.1.204

Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-sepharose affinity chromatography.

K R Westcott, D C La Porte, D R Storm
PMCID: PMC382906  PMID: 284333

Abstract

Partially purified adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from bovine brain cortex was fractionated into two separate forms by calcium-dependent regulatory protein (CDR)-Sepharose affinity chromatography. The major form of the enzyme, comprising approximately 80% of the applied activity, did not bind to the affinity column in the presence of Ca2+ and was insensitive to the CDR. Approximately 20% of adenylate cyclase activity was absorbed to CDR-Sepharose in the presence of Ca2+. This activity was stimulated by Ca2+ and CDR. This study directly demonstrates that brain cortex contains Ca2+-CDR-sensitive and -insensitive forms of adenylate cyclase and indicates that CDR-Sepharose may be a useful tool for purification of adenylate cyclase. The Ca2+ -stimulated adenylate cyclase was purified at least 55-fold with a 13% yield.

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Selected References

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

  1. Bradham L. S., Holt D. A., Sims M. The effect of Ca2+ on the adenyl cyclase of calf brain. Biochim Biophys Acta. 1970 Feb 24;201(2):250–260. doi: 10.1016/0304-4165(70)90299-0. [DOI] [PubMed] [Google Scholar]
  2. Brostrom C. O., Brostrom M. A., Wolff D. J. Calcium-dependent adenylate cyclase from rat cerebral cortex. Reversible activation by sodium fluoride. J Biol Chem. 1977 Aug 25;252(16):5677–5685. [PubMed] [Google Scholar]
  3. Brostrom C. O., Huang Y. C., Breckenridge B. M., Wolff D. J. Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc Natl Acad Sci U S A. 1975 Jan;72(1):64–68. doi: 10.1073/pnas.72.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brostrom M. A., Brostrom C. O., Breckenridge B. M., Wolff D. J. Regulation of adenylate cyclase from glial tumor cells by calcium and a calcium-binding protein. J Biol Chem. 1976 Aug 10;251(15):4744–4750. [PubMed] [Google Scholar]
  5. Cheung W. Y., Bradham L. S., Lynch T. J., Lin Y. M., Tallant E. A. Protein activator of cyclic 3':5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase. Biochem Biophys Res Commun. 1975 Oct 6;66(3):1055–1062. doi: 10.1016/0006-291x(75)90747-0. [DOI] [PubMed] [Google Scholar]
  6. Ho H. C., Wirch E., Stevens F. C., Wang J. H. Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein. J Biol Chem. 1977 Jan 10;252(1):43–50. [PubMed] [Google Scholar]
  7. Homcy C., Wrenn S., Haber E. Affinity purification of cardiac adenylate cyclase: dependence on prior hydrophobic resolution. Proc Natl Acad Sci U S A. 1978 Jan;75(1):59–63. doi: 10.1073/pnas.75.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johnson R. A., Sutherland E. W. Detergent-dispersed adenylate cyclase from rat brain. Effects of fluoride, cations, and chelators. J Biol Chem. 1973 Jul 25;248(14):5114–5121. [PubMed] [Google Scholar]
  9. Kakiuchi S., Yamazaki R., Teshima Y., Uenishi K. Regulation of nucleoside cyclic 3':5'-monophosphate phosphodiesterase activity from rat brain by a modulator and Ca2+. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3526–3530. doi: 10.1073/pnas.70.12.3526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Klee C. B., Krinks M. H. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978 Jan 10;17(1):120–126. doi: 10.1021/bi00594a017. [DOI] [PubMed] [Google Scholar]
  11. LaPorte D. C., Storm D. R. Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein. J Biol Chem. 1978 May 25;253(10):3374–3377. [PubMed] [Google Scholar]
  12. 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]
  13. Limbird L. E., Lefkowitz R. J. Resolution of beta-adrenergic receptor binding and adenylate cyclase activity by gel exclusion chromatography. J Biol Chem. 1977 Jan 25;252(2):799–802. [PubMed] [Google Scholar]
  14. Lin Y. M., Liu Y. P., Cheung W. Y. Cyclic 3':5'-nucleotide phosphodiesterase. Purification, characterization, and active form of the protein activator from bovine brain. J Biol Chem. 1974 Aug 10;249(15):4943–4954. [PubMed] [Google Scholar]
  15. Lynch T. J., Tallant E. A., Cheung W. Y. Ca++-dependent formation of brain adenylate cyclase-protein activator complex. Biochem Biophys Res Commun. 1976 Jan 26;68(2):616–625. doi: 10.1016/0006-291x(76)91190-6. [DOI] [PubMed] [Google Scholar]
  16. MacDonald I. A. Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100. Biochim Biophys Acta. 1975 Jul 27;397(1):244–253. doi: 10.1016/0005-2744(75)90197-7. [DOI] [PubMed] [Google Scholar]
  17. Naya-Vigne J., Johnson G. L., Bourne H. R., Coffino P. Complementation analysis of hormone-sensitive adenylate cyclase. Nature. 1978 Apr 20;272(5655):720–722. doi: 10.1038/272720a0. [DOI] [PubMed] [Google Scholar]
  18. Neer E. J. Size and detergent binding of adenylate cyclase from bovine cerebral cortex. J Biol Chem. 1978 Mar 10;253(5):1498–1502. [PubMed] [Google Scholar]
  19. Orly J., Schramm M. Coupling of catecholamine receptor from one cell with adenylate cyclase from another cell by cell fusion. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4410–4414. doi: 10.1073/pnas.73.12.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  21. Pfeuffer T., Helmreich E. J. Activation of pigeon erythrocyte membrane adenylate cyclase by guanylnucleotide analogues and separation of a nucleotide binding protein. J Biol Chem. 1975 Feb 10;250(3):867–876. [PubMed] [Google Scholar]
  22. Ross E. M., Gilman A. G. Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3715–3719. doi: 10.1073/pnas.74.9.3715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  24. Stellwagen E., Baker B. Proposed structure for brain adenylate cyclase purified using blue dextran-Sepharose chromatography. Nature. 1976 Jun 24;261(5562):719–720. doi: 10.1038/261719a0. [DOI] [PubMed] [Google Scholar]
  25. Takai K., Kurashina Y., Hayaishi O. Adenylate cyclase from Brevibacterium liquefaciens. Methods Enzymol. 1974;38:160–169. doi: 10.1016/0076-6879(74)38025-1. [DOI] [PubMed] [Google Scholar]
  26. Von Hungen K., Roberts S. Catecholamine and Ca 2+ activation of adenylate cyclase systems in synaptosomal fractions from rat cerebral cortex. Nat New Biol. 1973 Mar 14;242(115):58–60. doi: 10.1038/newbio242058a0. [DOI] [PubMed] [Google Scholar]
  27. Wallace R. W., Lynch T. J., Tallant E. A., Cheung W. Y. An endogenous inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Arch Biochem Biophys. 1978 Apr 30;187(2):328–334. doi: 10.1016/0003-9861(78)90042-5. [DOI] [PubMed] [Google Scholar]
  28. Wang J. H., Teo T. S., Ho H. C., Stevens F. C. Bovine heart protein activator of cyclic nucleotide phosphodiesterase. Adv Cyclic Nucleotide Res. 1975;5:179–194. [PubMed] [Google Scholar]
  29. Watterson D. M., Vanaman T. C. Affinity chromatography purification of a cyclic nucleotide phosphodiesterase using immobilized modulator protein, a troponin C-like protein from brain. Biochem Biophys Res Commun. 1976 Nov 8;73(1):40–46. doi: 10.1016/0006-291x(76)90494-0. [DOI] [PubMed] [Google Scholar]
  30. Wolff D. J., Brostrom C. O. Calcium-binding phosphoprotein from pig brain: identification as a calcium-dependent regulator of brain cyclic nucleotide phosphodiesterase. Arch Biochem Biophys. 1974 Jul;163(1):349–358. doi: 10.1016/0003-9861(74)90486-x. [DOI] [PubMed] [Google Scholar]

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