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. 1979 Oct;76(10):4903–4907. doi: 10.1073/pnas.76.10.4903

Sequential adsorption-electrophoresis: combined procedure for purification of calcium-dependent cyclic nucleotide phosphodiesterase.

R L Kincaid, M Vaughan
PMCID: PMC413046  PMID: 228270

Abstract

A procedure for combined sequential affinity adsorption-electrophoresis has been devised. Its use for the rapid purification of a calcium-dependent cyclic nucleotide phosphodiesterase from bovine brain in high yield is described. In this procedure, proteins bound to a solid phase of calcium-dependent regulatory protein (CDR) linked to Sepharose 4B were electrophoretically eluted, concentrated, and separated, thus avoiding the large losses in activity incurred during attempts to purify further the phosphodiesterase eluted by conventional means. The highly purified phosphodiesterase prepared by this method was stable for months at -60 degrees C in the presence of glycerol. It has a higher affinity for cyclic GMP than for cyclic AMP, and hydrolysis of both substrates is stimulated 5- to 6-fold by calcium plus CDR. Factors that influence adsorption of the enzyme to CDR-Sepharose and selection of optimal conditions for electrophoresis were investigated. Sequential adsorption-electrophoresis should be generally useful in the purification of macromolecules for which affinity adsorbents are available. The procedures described here could be directly applicable to the purification of proteins that, like the phosphodiesterase, interact with CDR.

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Selected References

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  1. Adelstein R. S., Conti M. A., Hathaway D. R., Klee C. B. Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase. J Biol Chem. 1978 Dec 10;253(23):8347–8350. [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. Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A. 1968 Oct;61(2):636–643. doi: 10.1073/pnas.61.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dabrowska R., Hartshorne D. J. A Ca2+-and modulator-dependent myosin light chain kinase from non-muscle cells. Biochem Biophys Res Commun. 1978 Dec 29;85(4):1352–1359. doi: 10.1016/0006-291x(78)91152-x. [DOI] [PubMed] [Google Scholar]
  5. Davis C. W., Kuo J. F. Purification and characterization of guanosine 3':5'-monophosphate-specific phosphodiesterase from guinea pig lung. J Biol Chem. 1977 Jun 25;252(12):4078–4084. [PubMed] [Google Scholar]
  6. Horejsí V., Kocourek J. Affinity electrophoresis: separation of phytohemagglutinins on O-glycosyl polyacrylamide gels. Methods Enzymol. 1974;34:178–181. doi: 10.1016/s0076-6879(74)34016-5. [DOI] [PubMed] [Google Scholar]
  7. Jean D. H., Albers R. W., Koval G. J. Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides. J Biol Chem. 1975 Feb 10;250(3):1035–1040. [PubMed] [Google Scholar]
  8. Kincaid R. L., Manganiello V. C., Vaughan M. Effects of spermine on activity and stability of calcium-dependent guanosine 3',5'-monophosphate phosphodiesterase. J Biol Chem. 1979 Jun 25;254(12):4970–4973. [PubMed] [Google Scholar]
  9. Klee C. B., Crouch T. H., Krinks M. H. Subunit structure and catalytic properties of bovine brain Ca2+-dependent cyclic nucleotide phosphodiesterase. Biochemistry. 1979 Feb 20;18(4):722–729. doi: 10.1021/bi00571a026. [DOI] [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. 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]
  12. Manganiello V., Vaughan M. An effect of insulin on cyclic adenosine 3':5'-monophosphate phosphodiesterase activity in fat cells. J Biol Chem. 1973 Oct 25;248(20):7164–7170. [PubMed] [Google Scholar]
  13. Miyake M., Daly J. W., Creveling C. R. Purification of calcium-dependent phosphodiesterases from rat cerebrum by affinity chromatography on activator protein-sepharose. Arch Biochem Biophys. 1977 May;181(1):39–45. doi: 10.1016/0003-9861(77)90481-7. [DOI] [PubMed] [Google Scholar]
  14. Morgan M. R., Kerr E. J., Dean P. D. Electrophoretic desorption: preparative elution of steroid specific antibodies from immunoadsorbents. J Steroid Biochem. 1978 Aug;9(8):767–770. doi: 10.1016/0022-4731(78)90197-8. [DOI] [PubMed] [Google Scholar]
  15. O'carra P., Barry S., Griffin T. Interfering and complicating adsorption effects in bioaffinity chromatography. Methods Enzymol. 1974;34:108–126. doi: 10.1016/s0076-6879(74)34011-6. [DOI] [PubMed] [Google Scholar]
  16. Parikh I., March S., Cuatercasas P. Topics in the methodology of substitution reactions with agarose. Methods Enzymol. 1974;34:77–102. doi: 10.1016/s0076-6879(74)34009-8. [DOI] [PubMed] [Google Scholar]
  17. Wallace R. W., Lynch T. J., Tallant E. A., Cheung W. Y. Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. J Biol Chem. 1979 Jan 25;254(2):377–382. [PubMed] [Google Scholar]
  18. 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]
  19. Westcott K. R., La Porte D. C., Storm D. R. Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-sepharose affinity chromatography. Proc Natl Acad Sci U S A. 1979 Jan;76(1):204–208. doi: 10.1073/pnas.76.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yazawa M., Yagi K. Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography. J Biochem. 1978 Nov;84(5):1259–1265. doi: 10.1093/oxfordjournals.jbchem.a132244. [DOI] [PubMed] [Google Scholar]

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