Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1980 Jun 1;85(3):916–923. doi: 10.1083/jcb.85.3.916

Brush-border calmodulin. A major component of the isolated microvillus core

PMCID: PMC2111444  PMID: 6893051

Abstract

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons.The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.

Full Text

The Full Text of this article is available as a PDF (770.4 KB).

Selected References

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

  1. Amphlett G. W., Vanaman T. C., Perry S. V. Effect of the troponin C-like protein from bovine brain (brain modulator protein) on the Mg2+-stimulated ATPase of skeletal muscle actinomyosin. FEBS Lett. 1976 Dec 15;72(1):163–168. doi: 10.1016/0014-5793(76)80836-8. [DOI] [PubMed] [Google Scholar]
  2. Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Booth A. G., Kenny A. J. A rapid method for the preparation of microvilli from rabbit kidney. Biochem J. 1974 Sep;142(3):575–581. doi: 10.1042/bj1420575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bretscher A., Weber K. Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy. J Cell Biol. 1978 Dec;79(3):839–845. doi: 10.1083/jcb.79.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bretscher A., Weber K. Purification of microvilli and an analysis of the protein components of the microfilament core bundle. Exp Cell Res. 1978 Oct 15;116(2):397–407. doi: 10.1016/0014-4827(78)90463-9. [DOI] [PubMed] [Google Scholar]
  6. Bretscher A., Weber K. Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci U S A. 1979 May;76(5):2321–2325. doi: 10.1073/pnas.76.5.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brostrom C. O., Huang Y. C., Breckenridge B. M., Wolff D. J. Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc Natl Acad Sci U S A. 1975 Jan;72(1):64–68. doi: 10.1073/pnas.72.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheung W. Y. Cyclic 3',5'-nucleotide phosphodiesterase. Evidence for and properties of a protein activator. J Biol Chem. 1971 May 10;246(9):2859–2869. [PubMed] [Google Scholar]
  9. Cheung W. Y., Lin Y. M. Purification and characterization of cyclic 3',5'-nucleotide phosphodiesterase from bovine brain. Methods Enzymol. 1974;38:223–239. [PubMed] [Google Scholar]
  10. Cohen P., Burchell A., Foulkes J. G., Cohen P. T., Vanaman T. C., Nairn C. Identification of the Ca2+-dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase. FEBS Lett. 1978 Aug 15;92(2):287–293. doi: 10.1016/0014-5793(78)80772-8. [DOI] [PubMed] [Google Scholar]
  11. Dabrowska R., Sherry J. M., Aromatorio D. K., Hartshorne D. J. Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry. 1978 Jan 24;17(2):253–258. doi: 10.1021/bi00595a010. [DOI] [PubMed] [Google Scholar]
  12. Dedman J. R., Brinkley B. R., Means A. R. Regulation of microfilaments and microtubules by calcium and cyclic AMP. Adv Cyclic Nucleotide Res. 1979;11:131–174. [PubMed] [Google Scholar]
  13. Drabikowski W., Kuznicki J., Grabarek Z. Similarity in Ca2+-induced changes between troponic-C and protein activator of 3':5'-cyclic nucleotide phosphodiesterase and their tryptic fragments. Biochim Biophys Acta. 1977 Nov 23;485(1):124–133. doi: 10.1016/0005-2744(77)90199-1. [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. Jarrett H. W., Penniston J. T. Partial purification of the Ca2+-Mg2+ ATPase activator from human erythrocytes: its similarity to the activator of 3':5' - cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1210–1216. doi: 10.1016/s0006-291x(77)80108-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lin Y. M., Liu Y. P., Cheung W. Y. Purification and characterization of a protein activator of cyclic nucleotide phosphodiesterase from bovine brain. Methods Enzymol. 1974;38:262–273. doi: 10.1016/0076-6879(74)38042-1. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Marcum J. M., Dedman J. R., Brinkley B. R., Means A. R. Control of microtubule assembly-disassembly by calcium-dependent regulator protein. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3771–3775. doi: 10.1073/pnas.75.8.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matsudaira P. T., Burgess D. R. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. doi: 10.1083/jcb.83.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mooseker M. S. Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium. J Cell Biol. 1976 Nov;71(2):417–433. doi: 10.1083/jcb.71.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mooseker M. S., Pollard T. D., Fujiwara K. Characterization and localization of myosin in the brush border of intestinal epithelial cells. J Cell Biol. 1978 Nov;79(2 Pt 1):444–453. doi: 10.1083/jcb.79.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mukherjee T. M., Staehelin L. A. The fine-structural organization of the brush border of intestinal epithelial cells. J Cell Sci. 1971 May;8(3):573–599. doi: 10.1242/jcs.8.3.573. [DOI] [PubMed] [Google Scholar]
  26. PORTZEHL H., CALDWELL P. C., RUEEGG J. C. THE DEPENDENCE OF CONTRACTION AND RELAXATION OF MUSCLE FIBRES FROM THE CRAB MAIA SQUINADO ON THE INTERNAL CONCENTRATION OF FREE CALCIUM IONS. Biochim Biophys Acta. 1964 May 25;79:581–591. doi: 10.1016/0926-6577(64)90224-4. [DOI] [PubMed] [Google Scholar]
  27. Rodewald R., Newman S. B., Karnovsky M. J. Contraction of isolated brush borders from the intestinal epithelium. J Cell Biol. 1976 Sep;70(3):541–554. doi: 10.1083/jcb.70.3.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Runge M. S., Hewgley P. B., Puett D., Williams R. C., Jr Cyclic nucleotide phosphodiesterase activity in 10-nm filaments and microtubule preparations from bovine brain. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2561–2565. doi: 10.1073/pnas.76.6.2561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Small J. V., Sobieszek A. Ca-regulation of mammalian smooth muscle actomyosin via a kinase-phosphatase-dependent phosphorylation and dephosphorylation of the 20 000-Mr light chain of myosin. Eur J Biochem. 1977 Jun 15;76(2):521–530. doi: 10.1111/j.1432-1033.1977.tb11622.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Szent-Györgyi A. G., Szentkiralyi E. M., Kendrick-Jonas J. The light chains of scallop myosin as regulatory subunits. J Mol Biol. 1973 Feb 25;74(2):179–203. doi: 10.1016/0022-2836(73)90106-x. [DOI] [PubMed] [Google Scholar]
  32. TAUSSKY H. H., SHORR E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953 Jun;202(2):675–685. [PubMed] [Google Scholar]
  33. Wieland T. Modification of actins by phallotoxins. Naturwissenschaften. 1977 Jun;64(6):303–309. doi: 10.1007/BF00446784. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES