Characterization Of The Sea-Urchin Egg Microtubule-Activated Atpase

Collins, Christine A.; Vallee, Richard B.

doi:10.1242/jcs.1986.supplement_5.13

Cited by 8 publications

(2 citation statements)

References 33 publications

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“…It is noteworthy that brain dynein, kinesin, and the 10S ATPase all have been reported to exhibit values of Kmt below 1.5 mg/ml tubulin (Fig. 1; Kuzentsov and Gelfand, 1986;Collins and Vallee, 1986b). Lye et al (1987) have reported the purification of a 20S…”

Section: Relationship Of the Brain Cytoplasmic Dynein To Other Microtmentioning

confidence: 98%

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

Shpetner¹,

Paschal²,

Vallee³

1988

The Journal of Cell Biology

144

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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 metavanadate (Ki = 5-10 ttM for the microtubulestimulated 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.

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Section: Relationship Of the Brain Cytoplasmic Dynein To Other Microtmentioning

confidence: 98%

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

Shpetner¹,

Paschal²,

Vallee³

1988

The Journal of Cell Biology

144

View full text Add to dashboard Cite

show abstract

“…To address the question of assembly competence and specificity of association, we performed experiments using an alternative assembly/disassembly procedure to remove proteins that were not integral components of the microtubule lattice. This procedure is based on taxol stabilization of microtubules followed by a dissociative treatment with high salt to remove microtubule-associated proteins (Vallee, 1982;Collins & Vallee, 1986;Dinsmore & Sloboda, 1987). In these experiments, procaryotic synthesis reaction crudes were mixed with [3H]Tyr45l-tubulin (an internal positive control) and purified by anion-exchange chromatography.…”

Section: Methodsmentioning

confidence: 99%

Expression of a human .alpha.-tubulin: properties of the isolated subunit

Yaffe¹,

Levison²,

Szasz³

et al. 1988

Biochemistry

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We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer.

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In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg

Rebhun

Palazzo

1988

Cell Motil. Cytoskeleton

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Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.

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Characterization Of The Sea-Urchin Egg Microtubule-Activated Atpase

Cited by 8 publications

References 33 publications

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

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

Expression of a human .alpha.-tubulin: properties of the isolated subunit

In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg

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