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. 1979 Aug;76(8):3708–3712. doi: 10.1073/pnas.76.8.3708

Reconstitution and purification by "transport specificity fractionation" of an ATP-dependent calcium transport component from synaptosome-derived vesicles.

D Papazian, H Rahamimoff, S M Goldin
PMCID: PMC383902  PMID: 158762

Abstract

A synaptosomal ATP-dependent Ca uptake system was reconstituted into artificial vesicles by a cholate dialysis procedure using an 80-fold excess of exogenous phospholipid. Under these conditions, most of these vesicles would be expected to have only one or, at most, a few membrane proteins. The vesicles containing an ATP-dependent Ca transport system were purified from the bulk of the preparation on density gradients by increasing their density by the ATP-dependent intravesicular precipitation of Ca oxalate; a approximately 100-fold purification resulted. The purified Ca-transporting vesicles contained two major protein components, of Mr 94,000 and 140,000 according to sodium dodecyl sulfate gel electrophoresis. These components are believed to be responsible for Ca transport in this synaptosome-derived membrane fraction.

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Selected References

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

  1. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  2. Baker P. F. Transport and metabolism of calcium ions in nerve. Prog Biophys Mol Biol. 1972;24:177–223. doi: 10.1016/0079-6107(72)90007-7. [DOI] [PubMed] [Google Scholar]
  3. Blaustein M. P., Ratzlaff R. W., Kendrick N. C., Schweitzer E. S. Calcium buffering in presynaptic nerve terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism. J Gen Physiol. 1978 Jul;72(1):15–41. doi: 10.1085/jgp.72.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blitz A. L., Fine R. E., Toselli P. A. Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum. J Cell Biol. 1977 Oct;75(1):135–147. doi: 10.1083/jcb.75.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drickamer L. K. The red cell membrane contains three different adenosine triphophatases. J Biol Chem. 1975 Mar 10;250(5):1952–1954. [PubMed] [Google Scholar]
  6. Endo M. Calcium release from the sarcoplasmic reticulum. Physiol Rev. 1977 Jan;57(1):71–108. doi: 10.1152/physrev.1977.57.1.71. [DOI] [PubMed] [Google Scholar]
  7. Epstein M., Racker E. Reconstitution of carbamylcholine-dependent sodium ion flux and desensitization of the acetylcholine receptor from Torpedo californica. J Biol Chem. 1978 Oct 10;253(19):6660–6662. [PubMed] [Google Scholar]
  8. Gasko O. D., Knowles A. F., Shertzer H. G., Suolinna E. M., Racker E. The use of ion-exchange resins for studying ion transport in biological systems. Anal Biochem. 1976 May 7;72:57–65. doi: 10.1016/0003-2697(76)90506-6. [DOI] [PubMed] [Google Scholar]
  9. Goldin S. M. Active transport of sodium and potassium ions by the sodium and potassium ion-activated adenosine triphosphatase from renal medulla. Reconstitution of the purified enzyme into a well defined in vitro transport system. J Biol Chem. 1977 Aug 25;252(16):5630–5642. [PubMed] [Google Scholar]
  10. Goldin S. M., Rhoden V. Reconstitution and "transport specificity fractionation" of the human erythrocyte glucose transport system. A new approach for identification and isolation of membrane transport proteins. J Biol Chem. 1978 Apr 25;253(8):2575–2583. [PubMed] [Google Scholar]
  11. HASSELBACH W., MAKINOSE M. [The calcium pump of the "relaxing granules" of muscle and its dependence on ATP-splitting]. Biochem Z. 1961;333:518–528. [PubMed] [Google Scholar]
  12. 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]
  13. Mannherz H. G., Goody R. S. Proteins of contractile systems. Annu Rev Biochem. 1976;45:427–465. doi: 10.1146/annurev.bi.45.070176.002235. [DOI] [PubMed] [Google Scholar]
  14. Racker E., Eytan E. A coupling factor from sarcoplasmic reticulum required for the translocation of Ca2+ ions in a reconstituted Ca2+ATPase pump. J Biol Chem. 1975 Sep 25;250(18):7533–7534. [PubMed] [Google Scholar]
  15. Racker E. Reconstitution of a calcium pump with phospholipids and a purified Ca ++ - adenosine triphosphatase from sacroplasmic reticulum. J Biol Chem. 1972 Dec 25;247(24):8198–8200. [PubMed] [Google Scholar]
  16. Rahamimoff H., Abramovitz E. Calcium transport in a vesicular membrane preparation from rat brain synaptosomes. FEBS Lett. 1978 May 15;89(2):223–226. doi: 10.1016/0014-5793(78)80222-1. [DOI] [PubMed] [Google Scholar]
  17. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  18. Simons T. J. The interaction of ATP-analogues possessing a blocked gamma-phosphate group with the sodium pump in human red cells. J Physiol. 1975 Jan;244(3):731–739. doi: 10.1113/jphysiol.1975.sp010822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tada M., Ohmori F., Yamada M., Abe H. Mechanism of the stimulation of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. Role of the 22,000-dalton protein. J Biol Chem. 1979 Jan 25;254(2):319–326. [PubMed] [Google Scholar]

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