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PMID: 2971069 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C).

The Journal of cell biology ·Vol. 107 ·No. 3 ·1988-09-00 ·Pages 1001-9

Shpetner HS, Paschal BM, Vallee RB

Abstract

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.

MeSH Terms
Adenosine Triphosphatases/analysis,antagonists & inhibitors,metabolism Animals Brain/enzymology,ultrastructure Centrifugation, Density Gradient Cytosol/enzymology Dyneins/analysis Microtubule-Associated Proteins/analysis,metabolism Microtubules/enzymology,metabolism Oxidation-Reduction Potassium Chloride/metabolism Substrate Specificity Tubulin/metabolism
Chemicals
Microtubule-Associated Proteins Tubulin Potassium Chloride Adenosine Triphosphatases Dyneins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Shpetner H S
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Paschal B M
Vallee R B
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51 references, click to expand
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.107.3.1001
Published
1988-09-00
Pages
1001-9
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2115295
Subset
IM
Grants
NIGMS NIH HHS · GM-26701 · United States
External Links
PubMed source DOI: 10.1083/jcb.107.3.1001 PMC Full Text View journal details
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