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. 1979 Feb 1;177(2):623–630. doi: 10.1042/bj1770623

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

Andrew C Newby ‡,*,†, Andreas Chrambach §
PMCID: PMC1186413  PMID: 435255

Abstract

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.

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Selected References

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

  1. Arinç E., Philpot M. Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits. J Biol Chem. 1976 Jun 10;251(11):3213–3220. [PubMed] [Google Scholar]
  2. Catt K. J., Dufau M. L. Peptide hormone receptors. Annu Rev Physiol. 1977;39:529–557. doi: 10.1146/annurev.ph.39.030177.002525. [DOI] [PubMed] [Google Scholar]
  3. Cuatrecasas P. Affinity chromatography and purification of the insulin receptor of liver cell membranes. Proc Natl Acad Sci U S A. 1972 May;69(5):1277–1281. doi: 10.1073/pnas.69.5.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davis K. A., Hatefi Y. Succinate dehydrogenase. I. Purification, molecular properties, and substructure. Biochemistry. 1971 Jun 22;10(13):2509–2516. doi: 10.1021/bi00789a014. [DOI] [PubMed] [Google Scholar]
  5. Evans W. H., Gurd J. W. Properties of a 5'-nucleotidase purified from mouse liver plasma membranes. Biochem J. 1973 May;133(1):189–199. doi: 10.1042/bj1330189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. FERGUSON K. A. STARCH-GEL ELECTROPHORESIS--APPLICATION TO THE CLASSIFICATION OF PITUITARY PROTEINS AND POLYPEPTIDES. Metabolism. 1964 Oct;13:SUPPL–SUPPL1002. doi: 10.1016/s0026-0495(64)80018-4. [DOI] [PubMed] [Google Scholar]
  7. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  8. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  9. Hokin L. E. Purification and properties of the (sodium + potassium)-activated adenosinetriphosphatase and reconstitution of sodium transport. Ann N Y Acad Sci. 1974;242(0):12–23. doi: 10.1111/j.1749-6632.1974.tb19075.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Imai Y., Sato R. A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits. Biochem Biophys Res Commun. 1974 Sep 9;60(1):8–14. doi: 10.1016/0006-291x(74)90164-8. [DOI] [PubMed] [Google Scholar]
  12. Johnson R. A., Garbers D. L., Pilkis S. J. Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase. J Supramol Struct. 1976;4(2):205–219. doi: 10.1002/jss.400040208. [DOI] [PubMed] [Google Scholar]
  13. Jorgensen P. L., Skou J. C., Solomonson L. P. Purification and characterization of (Na+ + K+)-ATPase. II. Preparation by zonal centrifugation of highly active (Na+ + K+)-ATPase from the outer medulla of rabbit kidneys. Biochim Biophys Acta. 1971 Apr 13;233(2):381–394. doi: 10.1016/0005-2736(71)90335-x. [DOI] [PubMed] [Google Scholar]
  14. Kagawa Y. Reconstitution of oxidative phosphorylation. Biochim Biophys Acta. 1972 Aug 4;265(3):297–338. [PubMed] [Google Scholar]
  15. MacLennan D. H. Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum. J Biol Chem. 1970 Sep 10;245(17):4508–4518. [PubMed] [Google Scholar]
  16. Marchesi V. T., Furthmayr H., Tomita M. The red cell membrane. Annu Rev Biochem. 1976;45:667–698. doi: 10.1146/annurev.bi.45.070176.003315. [DOI] [PubMed] [Google Scholar]
  17. Meunier J. C., Sealock R., Olsen R., Changeux J. P. Purification and properties of the cholinergic receptor protein from Electrophorus electricus electric tissue. Eur J Biochem. 1974 Jun 15;45(2):371–394. doi: 10.1111/j.1432-1033.1974.tb03563.x. [DOI] [PubMed] [Google Scholar]
  18. Neer E. J. The size of adenylate cyclase. J Biol Chem. 1974 Oct 25;249(20):6527–6531. [PubMed] [Google Scholar]
  19. Newby A. C., Rodbell M., Chrambach A. Adenylate cyclase in polyacrylamide gel electrophoresis: solubilized but active. Arch Biochem Biophys. 1978 Sep;190(1):109–117. doi: 10.1016/0003-9861(78)90257-6. [DOI] [PubMed] [Google Scholar]
  20. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  21. Oesterhelt D., Stoeckenius W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat New Biol. 1971 Sep 29;233(39):149–152. doi: 10.1038/newbio233149a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Queener S. F., Fleming J. W., Bell N. H. Solubilization of calcitonin-responsive renal cortical adenylate cyclase. J Biol Chem. 1975 Oct 10;250(19):7586–7592. [PubMed] [Google Scholar]
  24. Roy C., Rajerison R., Bockaert J., Jard S. Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes. J Biol Chem. 1975 Oct 10;250(19):7885–7893. [PubMed] [Google Scholar]
  25. Slavik M., Kartner N., Riordan J. R. Lectin-induced inhibition of plasma membrane 5'-nucleotidase: sensitivity of purified enzyme. Biochem Biophys Res Commun. 1977 Mar 21;75(2):342–349. doi: 10.1016/0006-291x(77)91048-8. [DOI] [PubMed] [Google Scholar]
  26. Swislocki N. I., Magnuson T., Tierney J. Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP. Arch Biochem Biophys. 1977 Feb;179(1):157–165. doi: 10.1016/0003-9861(77)90099-6. [DOI] [PubMed] [Google Scholar]
  27. Swislocki N. I., Tierney J. Solubilization, stabilization, and partial purification of brain adenylate cyclase from rat. Biochemistry. 1973 May 8;12(10):1862–1866. doi: 10.1021/bi00734a004. [DOI] [PubMed] [Google Scholar]
  28. Takai K., Kurashina Y., Suzuki-Hori C., Okamoto H., Hayaishi O. Adenylate cyclase from Brevibacterium liquefaciens. I. Purification, crystallization, and some properties. J Biol Chem. 1974 Mar 25;249(6):1965–1972. [PubMed] [Google Scholar]
  29. Tanford C., Reynolds J. A. Characterization of membrane proteins in detergent solutions. Biochim Biophys Acta. 1976 Oct 26;457(2):133–170. doi: 10.1016/0304-4157(76)90009-5. [DOI] [PubMed] [Google Scholar]
  30. Wasserman A. R. Purification of membrane proteins: chloroplast cytochromes f and b559. Methods Enzymol. 1974;32:406–422. doi: 10.1016/0076-6879(74)32040-x. [DOI] [PubMed] [Google Scholar]
  31. Welton A. F., Lad P. M., Newby A. C., Yamamura H., Nicosia S., Rodbell M. Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states. J Biol Chem. 1977 Sep 10;252(17):5947–5950. [PubMed] [Google Scholar]
  32. Welton A. F., Lad P. M., Newby A. C., Yamamura H., Nicosia S., Rodbell M. The characteristics of lubrol-solubilized adenylate cyclase from rat liver plasma membranes. Biochim Biophys Acta. 1978 Feb 10;522(2):625–639. doi: 10.1016/0005-2744(78)90093-1. [DOI] [PubMed] [Google Scholar]
  33. Widnell C. C. Purification of rat liver 5'-nucleotidase as a complex with sphingomyelin. Methods Enzymol. 1974;32:368–374. doi: 10.1016/0076-6879(74)32037-x. [DOI] [PubMed] [Google Scholar]
  34. van der Hoeven T. A., Coon M. J. Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from rabbit liver microsomes. J Biol Chem. 1974 Oct 10;249(19):6302–6310. [PubMed] [Google Scholar]

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