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. 1980 Oct 1;87(1):84–97. doi: 10.1083/jcb.87.1.84

Interactions of dynein arms with b subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphosphate

PMCID: PMC2110734  PMID: 6448256

Abstract

Tetrahymena 30S dynein was extracted with 0.5 M KCl and tested for retention of several functional properties associated wtih its in situ force-generating capacity. The dynein fraction will rebind to extracted outer doublets in the presence of Mg2+ to restore dynein arms. The arms attach at one end to the A subfiber and form bridges at the other end to the B subfiber of an adjacent doublet. Recombined arms retain an ATPase activity that remains coupled to potential generation of interdoublet sliding forces. To examine important aspects of the dynein- tubulin interaction that we presume are directly related to the dynein force-generating cross-bridge cycle, a simple and quantitative spectrophotometric assay was devised for monitoring the associations between isolated 30S dynein and the B subfiber. Utilizing this assay, the binding of dynein to B subfibers was found to be dependent upon divalent cations, saturating at 3 mM Mg2+. Micromolar concentrations of MgATP2- cause the release of dynein from the B subfiber; however, not all of the dynein bound under these conditions is released by ATP. ATP- insensitive dynein binding results from dynein interactions with non-B- tubule sites on outer-doublet and central-pair microtubules and from ATP-insensitive binding to sites on the B subfiber. Vanadate over a wide concentration range (10(-6)-10(-3) M) has no effect on the Mg2+- induced binding of dynein or its release by MgATP2-, and was used to inhibit secondary doublet disintegration in the suspensions. In the presence of 10 microM vanadate, dynein is maximally dissociated by MgATP2- concentrations greater than or equal to 1 microM with half- maximal release at 0.2 microM. These binding properties of isolated dynein arms closely resemble the cross-bridging behavior of in situ dynein arms reported previously, suggesting that quantitative studies such as those presented here may yield reliable information concerning the mechanism of force generation in dynein-microtubule motile systems. The results also suggest that vanadate may interact with an enzyme- product complex that has a low affinity for tubulin.

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Selected References

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  1. Amos L., Klug A. Arrangement of subunits in flagellar microtubules. J Cell Sci. 1974 May;14(3):523–549. doi: 10.1242/jcs.14.3.523. [DOI] [PubMed] [Google Scholar]
  2. Bagshaw C. R., Trentham D. R. The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction. Biochem J. 1974 Aug;141(2):331–349. doi: 10.1042/bj1410331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cantley L. C., Jr, Cantley L. G., Josephson L. A characterization of vanadate interactions with the (Na,K)-ATPase. Mechanistic and regulatory implications. J Biol Chem. 1978 Oct 25;253(20):7361–7368. [PubMed] [Google Scholar]
  5. GIBBONS I. R. STUDIES ON THE PROTEIN COMPONENTS OF CILIA FROM TETRAHYMENA PYRIFORMIS. Proc Natl Acad Sci U S A. 1963 Nov;50:1002–1010. doi: 10.1073/pnas.50.5.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gibbons B. H., Gibbons I. R. Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with KC1. Biochem Biophys Res Commun. 1976 Nov 8;73(1):1–6. doi: 10.1016/0006-291x(76)90488-5. [DOI] [PubMed] [Google Scholar]
  7. Gibbons B. H., Gibbons I. R. Properties of flagellar "rigor waves" formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm. J Cell Biol. 1974 Dec;63(3):970–985. doi: 10.1083/jcb.63.3.970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibbons I. R. Chemical dissection of cilia. Arch Biol (Liege) 1965;76(2):317–352. [PubMed] [Google Scholar]
  9. Gibbons I. R., Cosson M. P., Evans J. A., Gibbons B. H., Houck B., Martinson K. H., Sale W. S., Tang W. J. Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate. Proc Natl Acad Sci U S A. 1978 May;75(5):2220–2224. doi: 10.1073/pnas.75.5.2220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haimo L. T., Telzer B. R., Rosenbaum J. L. Dynein binds to and crossbridges cytoplasmic microtubules. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5759–5763. doi: 10.1073/pnas.76.11.5759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoshino M. Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication. Biochim Biophys Acta. 1975 Oct 22;403(2):544–553. doi: 10.1016/0005-2744(75)90083-2. [DOI] [PubMed] [Google Scholar]
  12. Hoshino M. Preparation and characterization of a dissociated 14-s form from 30-S dynein of Tetrahymena cilia. Biochim Biophys Acta. 1974 May 10;351(1):142–154. doi: 10.1016/0005-2795(74)90073-7. [DOI] [PubMed] [Google Scholar]
  13. Hoshino M. Tryptic fragmentation of 30-S dynein from Tetrahymena cilia. Biochim Biophys Acta. 1977 May 27;492(1):70–82. doi: 10.1016/0005-2795(77)90215-x. [DOI] [PubMed] [Google Scholar]
  14. Kobayashi T., Martensen T., Nath J., Flavin M. Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1313–1318. doi: 10.1016/0006-291x(78)91279-2. [DOI] [PubMed] [Google Scholar]
  15. Lymn R. W., Taylor E. W. Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry. 1971 Dec 7;10(25):4617–4624. doi: 10.1021/bi00801a004. [DOI] [PubMed] [Google Scholar]
  16. Marston S. B., Rodger C. D., Tregear R. T. Changes in muscle crossbridges when beta, gamma-imido-ATP binds to myosin. J Mol Biol. 1976 Jun 14;104(1):263–276. doi: 10.1016/0022-2836(76)90012-7. [DOI] [PubMed] [Google Scholar]
  17. Okuno M., Brokaw C. J. Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and P1. J Cell Sci. 1979 Aug;38:105–123. doi: 10.1242/jcs.38.1.105. [DOI] [PubMed] [Google Scholar]
  18. Okuno M. Inhibition and relaxation of sea urchin sperm flagella by vanadate. J Cell Biol. 1980 Jun;85(3):712–725. doi: 10.1083/jcb.85.3.712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Otokawa M. Inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis. Biochim Biophys Acta. 1973 Apr 5;292(3):834–836. doi: 10.1016/0005-2728(73)90030-3. [DOI] [PubMed] [Google Scholar]
  20. Penningroth S. M., Witman G. B. Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm. J Cell Biol. 1978 Dec;79(3):827–832. doi: 10.1083/jcb.79.3.827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raff E. C., Blum J. J. The fractionation of glycerinated cilia by adenosine triphosphate. J Biol Chem. 1969 Jan 25;244(2):366–376. [PubMed] [Google Scholar]
  22. Sale W. S., Gibbons I. R. Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella. J Cell Biol. 1979 Jul;82(1):291–298. doi: 10.1083/jcb.82.1.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sale W. S., Satir P. Direction of active sliding of microtubules in Tetrahymena cilia. Proc Natl Acad Sci U S A. 1977 May;74(5):2045–2049. doi: 10.1073/pnas.74.5.2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shimizu T., Kimura I. Effects of adenosine triphosphate on N-ethylmaleimide-induced modification of 30S dynein from Tetrahymena cilia. J Biochem. 1977 Jul;82(1):165–173. doi: 10.1093/oxfordjournals.jbchem.a131665. [DOI] [PubMed] [Google Scholar]
  25. Summers K. E., Gibbons I. R. Effects of trypsin digestion on flagellar structures and their relationship to motility. J Cell Biol. 1973 Sep;58(3):618–629. doi: 10.1083/jcb.58.3.618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Takahashi M., Tonomura Y. Binding of 30s dynein with the B-tubule of the outer doublet of axonemes from Tetrahymena pyriformis and adenosine triphosphate-induced dissociation of the complex. J Biochem. 1978 Dec;84(6):1339–1355. doi: 10.1093/oxfordjournals.jbchem.a132256. [DOI] [PubMed] [Google Scholar]
  28. Takahashi M., Tonomura Y. Kinetic properties of dynein ATPase from Tetrahymena pyriformis. The initial phosphate burst of dynein ATPase and its interaction with ATP analogs. J Biochem. 1979 Aug;86(2):413–423. doi: 10.1093/oxfordjournals.jbchem.a132540. [DOI] [PubMed] [Google Scholar]
  29. Trentham D. R., Bardsley R. G., Eccleston J. F., Weeds A. G. Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromyosin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ. Biochem J. 1972 Feb;126(3):635–644. doi: 10.1042/bj1260635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. VanEtten R. L., Waymack P. P., Rehkop D. M. Letter: Transition metal ion inhibition of enzyme-catalyzed phosphate ester displacement reactions. J Am Chem Soc. 1974 Oct 16;96(21):6782–6785. doi: 10.1021/ja00828a053. [DOI] [PubMed] [Google Scholar]
  31. Warner F. D., Mitchell D. R. Structural conformation of ciliary dynein arms and the generation of sliding forces in Tetrahymena cilia. J Cell Biol. 1978 Feb;76(2):261–277. doi: 10.1083/jcb.76.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Warner F. D., Zanetti N. C. Properties of microtubule sliding disintegration in isolated Tetrahymena cilia. J Cell Biol. 1980 Aug;86(2):436–445. doi: 10.1083/jcb.86.2.436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zanetti N. C., Mitchell D. R., Warner F. D. Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding. J Cell Biol. 1979 Mar;80(3):573–588. doi: 10.1083/jcb.80.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]

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