Advertisement

Histochemistry

, Volume 78, Issue 2, pp 181–194 | Cite as

The ATPase activity in brain microtubule preparations is membrane-associated

  • K. Prus
  • M. Wallin
Article

Summary

Microtubule protein prepared from bovine brain by a temperature-dependent assembly-disassembly procedure contained Mg2+-or Ca2+-stimulated ATPase activity. However, activity decreased with successive cycles of assembly-disassembly such that 15% of the Mg2+-stimulated and 31% of the Ca2+-stimulated activity of the second-cycle material remained after seven cycles. Microtubule preparations purified by three cycles of assembly-disassembly contained many membrane fragments and vesicles which were absent in microtubule preparations cycled eight times. Histochemistry and electron microscopy revealed that much of the activity is associated with the vesicles. Vesicles with an accumulation of lead phosphate deposits (indication of ATPase activity) were observed in high-speed pellets (150,000 g, 60 min) of microtubule-associated proteins. Most of the activity in the microtubule-associated protein preparations, but only a fraction of the total protein is pelleted. 53–78% of the ATPase activity, but only 6% of the total protein, is recovered in a microtubule-associated protein fraction eluted from phosphocellulose with 0.17 M NaCl. Polypeptides resolved on SDS polyacrylamide gradient gels have estimated molecular weights of 30,000–76,000. Electron micrographs of this material revealed short filaments, vesicles, and small ring-like structures. None of the inhibitors of possible contaminating ATPases affected the ATPase activity.

Keywords

ATPase Activity Protein Preparation Bovine Brain Successive Cycle Membrane Fragment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bhattacharyya B, Wolff J (1975) Membrane-bound tubulin in brain and thyroid tissue. J Biol Chem 250: 7639–7649Google Scholar
  2. Bhattacharyya B, Wolff J (1976) Polymerization of membrane tubulin. Nature 264: 576–577Google Scholar
  3. Bird MM (1976) Microtubule-synaptic vesicle associations in cultured rat spinal cord neurons. Cell Tissue Res 168: 101–115Google Scholar
  4. Borisy GG, Olmsted JB, Marcum JM, Allen C (1974) Microtubule assembly in vitro. Fed Proc 33: 167–174Google Scholar
  5. Burns RG, Pollard TD (1974) A dynein-like protein from brain. FEBS Lett 40: 274–280Google Scholar
  6. Daleo GR, Piras MM, Piras R (1974) The presence of phospholipids and diglyceride kinase activity in microtubules from different tissues. Biochem Biophys Res Commun 61: 1043–1050Google Scholar
  7. Feit H, Shay JW (1980) The assembly of tubulin into membranes. Biochem Biophys Res Commun 94: 324–331Google Scholar
  8. Franke WW (1971) Cytoplasmic microtubules linked to endoplasmic reticulum with crossbridges. Exp Cell Res 66: 486–489Google Scholar
  9. Gaskin F, Kramer SB, Cantor CR, Adelstein R, Shelanski ML (1974) A dynein-like protein associated with neurotubules. FEBS Lett 40: 281–286Google Scholar
  10. Gelfand VI, Gyoeva FK, Rosenblat VA, Shahina NA (1978) A new ATPase in cytoplasmic microtubule preparations. FEBS Lett 88: 197–200Google Scholar
  11. Gray EG (1975) Presynaptic microtubules and their association with synaptic vesicles. Proc R Soc Lond B 190: 369–372Google Scholar
  12. Järlfors U, Smith DS (1969) Association between synaptic vesicles and neurotubules. Nature 224: 710–711Google Scholar
  13. Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27: 137–138Google Scholar
  14. Lagnado JR, Kirazov EK (1975) Studies on the phosphorylation of brain microtubule protein and microtubule-associated phospholipids. In: Borgers M, DeBrabander M (eds) Microtubules and microtubule inhibitors. North Holland, Amsterdam, pp 127–140Google Scholar
  15. Larsson H, Wallin M, Edström A (1976) Induction of a sheet polymer of tubulin by Zn2+. Exp Cell Res 100: 104–110Google Scholar
  16. Larsson H, Wallin M, Edström A (1979) Some characteristics of ATPase activity in a brain microtubule protein preparation. J Neurochem 33: 1249–1258Google Scholar
  17. Ledig M, Ciesielski-Treska J, Cam Y, Montagnon D, Mandel P (1975) ATPase activity of neuroblastoma cells in culture. J Neurochem 25: 635–640Google Scholar
  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275Google Scholar
  19. Nath J, Flavin M (1978) A structural difference between cytoplasmic and membrane-bound tubulin of brain. FEBS Lett 95: 335–338Google Scholar
  20. O'Farrell PH (1975) High-resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021Google Scholar
  21. Prus K, Wallin M (1982) Microtubule-associated ATPase: fact or artifact? In: Weiss D (ed) Axoplasmic transport. Springer, Berlin Heidelberg New York, pp 91–98Google Scholar
  22. Raine CS, Ghetti B, Shelanski ML (1971) On the association between microtubules and mitochondria within axons. Brain Res 34: 389–393Google Scholar
  23. Senior AE (1973) The structure of mitochondrial ATPase. Biochim Biophys Acta 301: 249–277Google Scholar
  24. Sharp GA, Fitzsimons JTR, Kerkut GA (1980) Electron microscopic localization of an ATPase associated with microtubules in the cerebral cortex of the rat. Comp Biochem Physiol 66: 415–429Google Scholar
  25. Sherline P, Lee Y-C, Jacobs LS (1977) Binding of microtubules to pituitary secretory granules and secretory granule membranes. J Cell Biol 72: 380–389Google Scholar
  26. Sloboda RD, Dentler WL, Rosenbaum JL (1976) Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry 15: 4497–4505Google Scholar
  27. Smith DS, Järlfors U, Cameron BF (1975) Morphological evidence for the participation of microtubules in axonal transport. Ann NY Acad Sci 253: 472–506Google Scholar
  28. Smith RS (1980) The short term accumulation of axonally transported organelles in the region of localized lesions of single myelinated axons. J Neurocytol 9: 39–65Google Scholar
  29. Suprenant KA, Dentler WL (1982) Association between endocrine pancreatic secretory granules and in vitro assembled microtubules is dependent upon microtubule-associated proteins. J Cell Biol 93: 164–174Google Scholar
  30. Wachstein M, Meisel E (1957) Histochemistry of hepatic phosphatases at physiologic pH. Am J Clin Pathol 27: 13–23Google Scholar
  31. Webb BC (1979) An ATPase activity associated with brain microtubules. Arch Biochem Biophys 198: 292–303Google Scholar
  32. Weil-Malherbe H, Green RH (1951) The catalytical effect of molybdate on the hydrolysis of organic phosphate bonds. Biochem J 49: 286–292Google Scholar
  33. Weingarten MD, Lockwood AH, Hwo S-Y, Kirschner MW (1975) A protein factor essential for microtubule assembly. Proc Natl Acad Sci USA 72: 1858–1862Google Scholar
  34. White HD, Coughlin BA, Purich DL (1980) Adenosine triphosphatase activity of bovine brain microtubules. J Biol Chem 255: 486–491Google Scholar
  35. Zisapel N, Levi M, Gozes I (1980) Tubulin: an integral protein of mammalian synaptic vesicle membranes. J Neurochem 34: 26–32Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • K. Prus
    • 1
  • M. Wallin
    • 1
  1. 1.Department of ZoophysiologyUniversity of GöteborgGöteborgSweden

Personalised recommendations