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. 1983 Mar 1;96(3):768–775. doi: 10.1083/jcb.96.3.768

Structural and dynamic states of actin in the erythrocyte

JC Pinder, WB Gratzer
PMCID: PMC2112381  PMID: 6682109

Abstract

Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons show it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1x10(5) and 5x10(5) for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays fro actin on lysed whole cells. The concentration of monomeric actin at 0 degrees C is approximately 16 μg/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing (“pointed”) ends of free actin filaments. This suggests that these ends of the actin “protofilaments” in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to approximately 180 μg/ml of packed cells, which is nearly 70 percent of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250 μg/ml of cells, compared with 270 μg/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion.

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Selected References

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

  1. Anderson J. M., Tyler J. M. State of spectrin phosphorylation does not affect erythrocyte shape or spectrin binding to erythrocyte membranes. J Biol Chem. 1980 Feb 25;255(4):1259–1265. [PubMed] [Google Scholar]
  2. Atkinson M. A., Morrow J. S., Marchesi V. T. The polymeric state of actin in the human erythrocyte cytoskeleton. J Cell Biochem. 1982;18(4):493–505. doi: 10.1002/jcb.1982.240180410. [DOI] [PubMed] [Google Scholar]
  3. Bennett V., Branton D. Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin. J Biol Chem. 1977 Apr 25;252(8):2753–2763. [PubMed] [Google Scholar]
  4. Bennett V. Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin. J Biol Chem. 1978 Apr 10;253(7):2292–2299. [PubMed] [Google Scholar]
  5. Birchmeier W., Singer S. J. On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. J Cell Biol. 1977 Jun;73(3):647–659. doi: 10.1083/jcb.73.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blikstad I., Markey F., Carlsson L., Persson T., Lindberg U. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell. 1978 Nov;15(3):935–943. doi: 10.1016/0092-8674(78)90277-5. [DOI] [PubMed] [Google Scholar]
  7. Brenner S. L., Korn E. D. Spectrin/actin complex isolated from sheep erythrocytes accelerates actin polymerization by simple nucleation. Evidence for oligomeric actin in the erythrocyte cytoskeleton. J Biol Chem. 1980 Feb 25;255(4):1670–1676. [PubMed] [Google Scholar]
  8. Brenner S. L., Korn E. D. The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization. J Biol Chem. 1980 Feb 10;255(3):841–844. [PubMed] [Google Scholar]
  9. Brown S. S., Spudich J. A. Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol. 1979 Dec;83(3):657–662. doi: 10.1083/jcb.83.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen C. M., Branton D. The role of spectrin in erythrocyte membrane-stimulated actin polymerisation. Nature. 1979 May 10;279(5709):163–165. doi: 10.1038/279163a0. [DOI] [PubMed] [Google Scholar]
  11. Cohen C. M., Tyler J. M., Branton D. Spectrin-actin associations studied by electron microscopy of shadowed preparations. Cell. 1980 Oct;21(3):875–883. doi: 10.1016/0092-8674(80)90451-1. [DOI] [PubMed] [Google Scholar]
  12. Cooke R. The role of the bound nucleotide in the polymerization of actin. Biochemistry. 1975 Jul 15;14(14):3250–3256. doi: 10.1021/bi00685a035. [DOI] [PubMed] [Google Scholar]
  13. Dunbar J. C., Ralston G. B. The incorporation of 32 P into spectrin aggregates following incubation of erythrocytes in 32 P-labelled inorganic phosphate. Biochim Biophys Acta. 1978 Jul 4;510(2):283–291. doi: 10.1016/0005-2736(78)90028-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Flanagan M. D., Lin S. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J Biol Chem. 1980 Feb 10;255(3):835–838. [PubMed] [Google Scholar]
  16. Garrels J. I., Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976 Dec;9(4 Pt 2):793–805. doi: 10.1016/0092-8674(76)90142-2. [DOI] [PubMed] [Google Scholar]
  17. Goodman S. R., Weidner S. A. Binding of spectrin alpha 2-beta 2 tetramers to human erythrocyte membranes. J Biol Chem. 1980 Sep 10;255(17):8082–8086. [PubMed] [Google Scholar]
  18. Gordon D. J., Yang Y. Z., Korn E. D. Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin. J Biol Chem. 1976 Dec 10;251(23):7474–7479. [PubMed] [Google Scholar]
  19. Gratzer W. B. The red cell membrane and its cytoskeleton. Biochem J. 1981 Jul 15;198(1):1–8. doi: 10.1042/bj1980001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Howard T. H., Lin S. Specific interaction of cytochalasins with muscle and platelet actin filaments in vitro. J Supramol Struct. 1979;11(3):283–293. doi: 10.1002/jss.400110303. [DOI] [PubMed] [Google Scholar]
  21. Jaenicke L. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal Biochem. 1974 Oct;61(2):623–627. doi: 10.1016/0003-2697(74)90429-1. [DOI] [PubMed] [Google Scholar]
  22. KASAI M., ASAKURA S., OOSAWA F. The G-F equilibrium in actin solutions under various conditions. Biochim Biophys Acta. 1962 Feb 12;57:13–21. doi: 10.1016/0006-3002(62)91072-7. [DOI] [PubMed] [Google Scholar]
  23. Koppel D. E., Sheetz M. P., Schindler M. Matrix control of protein diffusion in biological membranes. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3576–3580. doi: 10.1073/pnas.78.6.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kouyama T., Mihashi K. Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem. 1981;114(1):33–38. [PubMed] [Google Scholar]
  25. 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]
  26. Lehrer S. S., Kerwar G. Intrinsic fluorescence of actin. Biochemistry. 1972 Mar 28;11(7):1211–1217. doi: 10.1021/bi00757a015. [DOI] [PubMed] [Google Scholar]
  27. Lin D. C., Lin S. High affinity binding of [3H]dihydrocytochalasin B to peripheral membrane proteins related to the control of cell shape in the human red cell. J Biol Chem. 1978 Mar 10;253(5):1415–1419. [PubMed] [Google Scholar]
  28. Lin D. C. Spectrin-4.1-actin complex of the human erythrocyte: molecular basis of its ability to bind cytochalasins with high-affinity and to accelerate actin polymerization in vitro. J Supramol Struct Cell Biochem. 1981;15(2):129–138. doi: 10.1002/jsscb.1981.380150204. [DOI] [PubMed] [Google Scholar]
  29. Liu S. C., Palek J. Metabolic dependence of protein arrangement in human erythrocyte membranes. II. Crosslinking of major proteins in ghosts from fresh and ATP-depleted red cells. Blood. 1979 Nov;54(5):1117–1130. [PubMed] [Google Scholar]
  30. Lundin A., Richardsson A., Thore A. Continous monitoring of ATP-converting reactions by purified firefly luciferase. Anal Biochem. 1976 Oct;75(2):611–620. doi: 10.1016/0003-2697(76)90116-0. [DOI] [PubMed] [Google Scholar]
  31. MacLean-Fletcher S., Pollard T. D. Mechanism of action of cytochalasin B on actin. Cell. 1980 Jun;20(2):329–341. doi: 10.1016/0092-8674(80)90619-4. [DOI] [PubMed] [Google Scholar]
  32. Marchesi V. T., Palade G. E. Inactivation of adenosine triphosphatase and disruption of red cell membrances by trypsin: protective effect of adenosine triphosphate. Proc Natl Acad Sci U S A. 1967 Sep;58(3):991–995. doi: 10.1073/pnas.58.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. NAKAO M., NAKAO T., YAMAZOE S., YOSHIKAWA H. Adenosine triphosphate and shape of erythrocytes. J Biochem. 1961 Jun;49:487–492. doi: 10.1093/oxfordjournals.jbchem.a127333. [DOI] [PubMed] [Google Scholar]
  34. Nakashima K., Beutler E. Comparison of structure and function of human erythrocyte and human muscle actin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):935–938. doi: 10.1073/pnas.76.2.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. OHNISHI T. Extraction of actin- and myosin-like proteins from erythrocyte membrane. J Biochem. 1962 Oct;52:307–308. doi: 10.1093/oxfordjournals.jbchem.a127620. [DOI] [PubMed] [Google Scholar]
  36. Patel V. P., Fairbanks G. Spectrin phosphorylation and shape change of human erythrocyte ghosts. J Cell Biol. 1981 Feb;88(2):430–440. doi: 10.1083/jcb.88.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pinder J. C., Bray D., Gratzer W. B. Control of interaction of spectrin and actin by phosphorylation. Nature. 1977 Dec 22;270(5639):752–754. doi: 10.1038/270752a0. [DOI] [PubMed] [Google Scholar]
  38. Pinder J. C., Clark S. E., Baines A. J., Morris E., Gratzer W. B. The construction of the red cell cytoskeleton. Prog Clin Biol Res. 1981;55:343–361. [PubMed] [Google Scholar]
  39. Pinder J. C., Gratzer W. B. Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative. Biochemistry. 1982 Sep 28;21(20):4886–4890. doi: 10.1021/bi00263a009. [DOI] [PubMed] [Google Scholar]
  40. Pinder J. C., Ungewickell E., Bray D., Gratzer W. B. The spectrin-actin complex and erythrocyte shape. J Supramol Struct. 1978;8(4):439–445. doi: 10.1002/jss.400080406. [DOI] [PubMed] [Google Scholar]
  41. Pinder J. C., Ungewickell E., Calvert R., Morris E., Gratzer W. B. Polymerisation of G-actin by spectrin preparations: identification of the active constituent. FEBS Lett. 1979 Aug 15;104(2):396–400. doi: 10.1016/0014-5793(79)80861-3. [DOI] [PubMed] [Google Scholar]
  42. Pollard T. D., Mooseker M. S. Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981 Mar;88(3):654–659. doi: 10.1083/jcb.88.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Potter J. D. The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils. Arch Biochem Biophys. 1974 Jun;162(2):436–441. doi: 10.1016/0003-9861(74)90202-1. [DOI] [PubMed] [Google Scholar]
  44. Price P. A., Liu T. Y., Stein W. H., Moore S. Properties of chromatographically purified bovine pancreatic deoxyribonuclease. J Biol Chem. 1969 Feb 10;244(3):917–923. [PubMed] [Google Scholar]
  45. Schindler M., Koppel D. E., Sheetz M. P. Modulation of membrane protein lateral mobility by polyphosphates and polyamines. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1457–1461. doi: 10.1073/pnas.77.3.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sheetz M. P., Casaly J. 2,3-Diphosphoglycerate and ATP dissociate erythrocyte membrane skeletons. J Biol Chem. 1980 Oct 25;255(20):9955–9960. [PubMed] [Google Scholar]
  47. Sheetz M. P. Integral membrane protein interaction with Triton cytoskeletons of erythrocytes. Biochim Biophys Acta. 1979 Oct 19;557(1):122–134. doi: 10.1016/0005-2736(79)90095-6. [DOI] [PubMed] [Google Scholar]
  48. Sheetz M. P., Painter R. G., Singer S. J. Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells. Biochemistry. 1976 Oct 5;15(20):4486–4492. doi: 10.1021/bi00665a024. [DOI] [PubMed] [Google Scholar]
  49. Sheetz M. P., Singer S. J. On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex. J Cell Biol. 1977 Jun;73(3):638–646. doi: 10.1083/jcb.73.3.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  51. Steck T. L. The organization of proteins in the human red blood cell membrane. A review. J Cell Biol. 1974 Jul;62(1):1–19. doi: 10.1083/jcb.62.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tilney L. G., Detmers P. Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ. J Cell Biol. 1975 Sep;66(3):508–520. doi: 10.1083/jcb.66.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tyler J. M., Reinhardt B. N., Branton D. Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin. J Biol Chem. 1980 Jul 25;255(14):7034–7039. [PubMed] [Google Scholar]
  54. Ungewickell E., Bennett P. M., Calvert R., Ohanian V., Gratzer W. B. In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte. Nature. 1979 Aug 30;280(5725):811–814. doi: 10.1038/280811a0. [DOI] [PubMed] [Google Scholar]
  55. Ungewickell E., Gratzer W. Self-association of human spectrin. A thermodynamic and kinetic study. Eur J Biochem. 1978 Aug 1;88(2):379–385. doi: 10.1111/j.1432-1033.1978.tb12459.x. [DOI] [PubMed] [Google Scholar]
  56. Vandekerckhove J., Weber K. At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J Mol Biol. 1978 Dec 25;126(4):783–802. doi: 10.1016/0022-2836(78)90020-7. [DOI] [PubMed] [Google Scholar]
  57. Wang K., Richards F. M. Reaction of dimethyl-3,3'-dithiobispropionimidate with intact human erythrocytes. Cross-linking of membrane proteins and hemoglobin. J Biol Chem. 1975 Aug 25;250(16):6622–6626. [PubMed] [Google Scholar]
  58. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]

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