The relationship between cobalt binding to tubulin and the stimulation of assembly.

Himes, Richard H.; Lee, Y C; Eagle, Geoffrey R.; Haskins, Kathryn; Bäbler, S.; Ellermeier, Jeremy R.

doi:10.1016/s0021-9258(19)83855-3

Cited by 16 publications

(4 citation statements)

References 18 publications

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“…When tubulin was incubated with 2 X KT4 M CoCl2 in 20 mM Pipes and 1 X 10-4 M GTP, pH 7.0, buffer, 0.8 atom of cobalt was incorporated per tubulin dimer. This result is similar to that reported by Himes et al (1982) of 0.9-1.3 mol of tightly bound cobalt per mole of tubulin dimer. The consistent incorporation of 0.8 mol of cobalt per mole of tubulin was observed only if the incubation was carried out by passage through a Sephadex G-25 column equilibrated with the appropriate buffer as described under Materials and Methods.…”

Section: Resultssupporting

confidence: 92%

“…Tubulin-Cobalt Complex. Cobalt can form a strong complex with tubulin (Himes et al, 1982;Jemiolo & Grisham, 1982;Buttlaire et al, 1980;Eagle et al, 1983), replacing the Mg2+ at the exchangeable GTP site (Jemiolo & Grisham, 1982). The present studies show that the ability of cobalt to be incorporated depends on the conditions employed and the state of liganding of tubulin.…”

Section: Discussionmentioning

confidence: 62%

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Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin

Ward¹,

Timasheff²

1988

Biochemistry

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The high-affinity metal divalent cation Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid (tubulin-ANS) complexes. While pure native tubulin readily incorporated 0.8 atom of Co2+ per tubulin alpha-beta dimer, incorporation was reduced to 0.4 atom of Co2+ per mole of tubulin when it was complexed with colchicine, indicating that the conformational change induced in tubulin by the binding of colchicine leads to a reduced accessibility of the divalent cation binding site linked to the E site without necessarily changing the intrinsic binding constant. The fluorescence emission spectra of tubulin-bound colchicine, allocolchicine, and ANS displayed a strong overlap with the Co2+ absorption spectrum, identifying these as adequate donor-acceptor pairs. Fluorescence energy-transfer measurements were carried out between tubulin-bound colchicine (or allocolchicine) and ANS as donors and tubulin-complexed Co2+ as acceptor. It was found that the distance between the ANS and the high-affinity divalent cation binding sites is greater than 28 A, while that between the colchicine and the divalent cation binding sites is greater than 24 A.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 62%

Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin

Ward¹,

Timasheff²

1988

Biochemistry

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“…The interaction of tubulin with Zn2+ is similar to its interaction with Co2+ (Himes et al, 1982). Both cations reduce the critical protein concentration necessary for assembly and induce assembly at low protein and cation concentrations.…”

Section: Discussionmentioning

confidence: 91%

“…Zn2+ does appear to bind to more sites with high-affinity than does Co2+. In the presence of GTP, Co2+ binds to one or two sites with a Kd of about 1 juM (Himes et al, 1982) whereas Zn2+ binds to three such sites. One Co2+ is bound to tubulin after incubation with the cation in the absence of GTP followed by treatment with phosphocellulose; as many as seven Zn2+ ions are bound after a similar treatment.…”

Section: Discussionmentioning

confidence: 99%

Tubulin-zinc interactions: binding and polymerizaton studies

Eagle¹,

Zombola²,

Himes³

1983

Biochemistry

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The binding of Zn2+ to tubulin and the ability of this cation to promote the polymorphic assembly of the protein were examined. Equilibrium binding showed the existence of more than 60 potential Zn2+ binding sites on the dimer, including a number of high-affinity sites. The number of high-affinity sites, estimated by using a standard amount of phosphocellulose to remove more weakly bound Zn2+, reached a maximum of 6-7.5 with increasing levels of Zn2+ in the incubation solution. The number also increased with time of incubation at a single Zn2+ concentration. It is suggested that tubulin is slowly denatured in the presence of Zn2+, exposing more binding sites. Cu+ and Cd2+ were effective inhibitors of Zn2+ binding; Mg2+, Mn2+, and Co2+ were much less effective, and Ca2+ was without effect. Zn2+ does not replace the tightly bound Mg2+. GTP reduces the amount of Zn2+ binding under equilibrium conditions and the amount bound to high-affinity sites. Zinc-induced protofilament sheets are produced at a Zn2+/tubulin ratio of 5 in the presence of 0.5 mM GTP, conditions where about two to three Zn2+ ions would be bound to the dimer. At higher GTP concentrations, less assembly occurred, and the products were narrower sheets and microtubules. Zn2+-tubulin, isolated from phosphocellulose, will not assemble unless Mg2+ and dimethyl sulfoxide (Me2SO) or more Zn2+ is added. Broad protofilament sheets, formed from Zn2+-tubulin in the presence of Mg2+ and Me2SO, contain slightly more than one Zn2+ per dimer. It is concluded that Zn2+ stimulates tubulin assembly by binding directly to the protein, not via a ZnGTP complex.

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Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds

Webster

Oxford

1996

J. Cell. Biochem.

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alpha-tubulin subunits within microtubules (MTs) can be post-translationally detyrosinated by a tubulin-specific carboxypeptidase (TCP) activity to form biochemically distinct MTs. Attempts to characterize and purify TCP have suffered from the inability to detect low levels of activity and to distinguish TCP from other, competing enzyme activities. We recently developed an assay for TCP [Webster et al. (1992) Biochemistry 31:5849] that uses taxol-stabilized MTs as the substrate. In this study, we exploited the increased sensitivity and specificity of this new assay to explore the effects of various agents that might act to either stimulate or inhibit this enzyme in vitro. We tested a variety of both monovalent and divalent cations for their ability to affect TCP, and tested whether the cations were affecting the enzyme, the substrate, or both. We found that TCP displayed salt-sensitive binding to MTs, characteristic of other, more well characterized MT-associated proteins. While both calcium and magnesium stimulated TCP activity over a narrow concentration range (2-10 mM), they inhibited activity at higher concentrations. Other divalent cations tested, including zinc, copper, and cobalt, inhibited TCP at virtually all concentrations tested, but to different levels (zinc > copper > cobalt). Most of the zinc-induced TCP inhibition was attributed to the interference with the normal binding of TCP to MTs. In addition, we examined the involvement of free sulfhydryl groups (which are important for the activities of many types of enzymes) in TCP activity by the addition of sulfhydryl-modifying compounds during the assay, and found that their addition reduced TCP activity mainly (but not solely) by their action on the extract that contained the TCP. Finally, we tested the ability of DL-benzylsuccinic acid, a potent inhibitor of carboxypeptidase A, to inhibit TCP. While carboxypeptidase A has been found, in other studies, to be inhibited by micromolar concentrations, TCP was affected only at concentrations above 20 mM, adding another proof that carboxypeptidase A and TCP are distinct enzyme activities.

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The relationship between cobalt binding to tubulin and the stimulation of assembly.

Cited by 16 publications

References 18 publications

Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin

Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin

Tubulin-zinc interactions: binding and polymerizaton studies

Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds

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