Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1980 Apr 15;188(1):47–54. doi: 10.1042/bj1880047

Reconstitution of the Ca2+-transport system of human erythrocytes.

K Gietzen, S Seiler, S Fleischer, H U Wolf
PMCID: PMC1162535  PMID: 6105871

Abstract

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte 'ghosts' was solubilized and reconstituted to form membranous vesicles capable of energized Ca2+ accumulation. The erythrocyte 'ghosts' for this purpose were prepared by using isoosmotic freeze-haemolysis in the presence of Tween 20 and proteinase inhibitors to stabilize the preparation. The reconstitution procedure is similar to that developed by Meissner & Fleischer [(1974) J. Biol. Chem. 249, 302-309] for skeletal-muscle sarcoplasmic-reticulum in that: (1) deoxycholate is used for the solubilization of the membrane; (2) controlled dialysis at near room temperature, rather than 0 degree C, is required in order to obtain a functional preparation capable of Ca2+ accumulation; and (3) membrane vesicles can be reassembled with protein/lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original membrane.

Full text

PDF
53

Images in this article

50-1PLATE 1
on p.50-1

Selected References

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

  1. Arnold A., Wolf H. U., Ackermann B. P., Bader H. An automated continuous assay of membrane-bound and solube ATPases and related enzymes. Anal Biochem. 1976 Mar;71(1):209–213. doi: 10.1016/0003-2697(76)90029-4. [DOI] [PubMed] [Google Scholar]
  2. Gopinath R. M., Vincenzi F. F. Phosphodiesterase protein activator mimics red blood cell cytoplasmic activator of (Ca2+-Mg2+)ATPase. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1203–1209. doi: 10.1016/s0006-291x(77)80107-1. [DOI] [PubMed] [Google Scholar]
  3. Harrison D. G., Long C. The calcium content of human erythrocytes. J Physiol. 1968 Dec;199(2):367–381. doi: 10.1113/jphysiol.1968.sp008658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Horton C. R., Cole W. Q. Depressed (Ca++)-transport ATPase in cystic fibrosis erythrocytes. Biochem Biophys Res Commun. 1970 Aug 11;40(3):505–509. doi: 10.1016/0006-291x(70)90930-7. [DOI] [PubMed] [Google Scholar]
  5. Jarrett H. W., Penniston J. T. Purification of the Ca2+-stimulated ATPase activator from human erythrocytes. Its membership in the class of Ca2+-binding modulator proteins. J Biol Chem. 1978 Jul 10;253(13):4676–4682. [PubMed] [Google Scholar]
  6. Luthra M. G., Hildenbrandt G. R., Hanahan D. J. Studies on an activator of the (Ca2+ plus Mg2+)-ATPase of human erythrocyte membranes. Biochim Biophys Acta. 1976 Jan 8;419(1):164–179. doi: 10.1016/0005-2736(76)90380-1. [DOI] [PubMed] [Google Scholar]
  7. Meissner G., Conner G. E., Fleischer S. Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca 2+ -pump and Ca 2+ -binding proteins. Biochim Biophys Acta. 1973 Mar 16;298(2):246–269. doi: 10.1016/0005-2736(73)90355-6. [DOI] [PubMed] [Google Scholar]
  8. Meissner G., Fleischer S. Characterization of sarcoplasmic reticulum from skeletal muscle. Biochim Biophys Acta. 1971 Aug 13;241(2):356–378. doi: 10.1016/0005-2736(71)90036-8. [DOI] [PubMed] [Google Scholar]
  9. Meissner G., Fleischer S. Dissociation and reconstitution of functional sarcoplasmic reticulum vesicles. J Biol Chem. 1974 Jan 10;249(1):302–309. [PubMed] [Google Scholar]
  10. Meissner G. Isolation and characterization of two types of sarcoplasmic reticulum vesicles. Biochim Biophys Acta. 1975 Apr 21;389(1):51–68. doi: 10.1016/0005-2736(75)90385-5. [DOI] [PubMed] [Google Scholar]
  11. Peterson S. W., Ronner P., Carafoli E. Partial purification and reconstitution of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes. Arch Biochem Biophys. 1978 Feb;186(1):202–210. doi: 10.1016/0003-9861(78)90481-2. [DOI] [PubMed] [Google Scholar]
  12. Quist E. E., Roufogalis B. D. Association of (Ca + Mg)-ATPase activity with ATP-dependent Ca uptake in vesicles prepared from human erythrocytes. J Supramol Struct. 1977;6(3):375–381. doi: 10.1002/jss.400060310. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. SINGLETON W. S., GRAY M. S., BROWN M. L., WHITE J. L. CHROMATOGRAPHICALLY HOMOGENEOUS LECITHIN FROM EGG PHOSPHOLIPIDS. J Am Oil Chem Soc. 1965 Jan;42:53–56. doi: 10.1007/BF02558256. [DOI] [PubMed] [Google Scholar]
  15. Scarpa A., Inesi G. Ionophore mediated equilibration of calcium ion gradients in fragmented-sarcoplasmic reticulum. FEBS Lett. 1972 May 15;22(3):273–276. doi: 10.1016/0014-5793(72)80248-5. [DOI] [PubMed] [Google Scholar]
  16. Schatzmann H. J. Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells. J Physiol. 1973 Dec;235(2):551–569. doi: 10.1113/jphysiol.1973.sp010403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wolf H. U., Dieckvoss G., Lichtner R. Purification and properties of high-affinity Ca2+-ATPase of human erythrocyte membranes. Acta Biol Med Ger. 1977;36(5-6):847–858. [PubMed] [Google Scholar]
  18. Wolf H. U., Gietzen K. Proceedings: The solubilization of high-affinity Ca-2+-ATPase of human erythrocyte membranes. Hoppe Seylers Z Physiol Chem. 1974 Oct;355(10):1272–1272. [PubMed] [Google Scholar]
  19. Wolf H. U. Studies on a Ca 2+ -dependent ATPase of human erythrocyte membranes. Effects of Ca 2+ and H + . Biochim Biophys Acta. 1972 May 9;266(2):361–375. doi: 10.1016/0005-2736(72)90094-6. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES