Reactivation <i>in vitro</i> of zinc-requiring apo-enzymes by rat liver zinc-thionein

Udom, Albert; Brady, F.

doi:10.1042/bj1870329

Cited by 228 publications

(81 citation statements)

References 43 publications

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“…Specifically, the rate constant for zinc transfer from MT to apo-SDH is three orders of magnitude less than that of about 20,000 M Ϫ1 ⅐s Ϫ1 estimated for apo-SDH and free zinc ions. In the few other enzyme systems studied (27), MT also failed to accelerate the reconstitution of the apoenzyme, as might have been expected if MT had insertase-like enzymatic activity such as, e.g., ferrochelatase, the enzyme that inserts iron into protoporphyrin IX. At best, the rate of zinc transfer is as rapid as that of free zinc ions in the case of apo-carbonic anhydrase (17).…”

Section: Discussionmentioning

confidence: 97%

The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase

Jiang

Maret

Vallée

1998

Proc. Natl. Acad. Sci. U.S.A.

284

207

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The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four.65 Znlabeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.The possible functional implications of the zinc cluster structure of metallothionein (MT) have been the subject of much speculation (1-3). In contrast with other zinc proteins, MT is remarkable in that it binds zinc with high thermodynamic stability [K d ϭ 1.4 ϫ 10 Ϫ13 M at pH 7.0 for human MT (4)] while exhibiting a kinetic lability that results in facile zinc exchange reactions (5). This unusual combination appears to be a characteristic property of the clusters and, likely, a critical element in their hypothetical function to ''[provide] zinc where and when needed and for whatever role'' (6). Thus, in MT the protein plays a role in the biological function of zinc, a paradigm quite different from that in most other zinc proteins where zinc plays a role in the biological function of the protein. The tight binding of zinc to MT raises questions of how it is released and whether or not the release is controlled. In this regard, we have identified glutathione disulfide (GSSG) as a cellular ligand that reacts with MT and mobilizes zinc, resulting in the suggestion that the zinc content of MT is linked to the redox state of glutathione in the cell in such a manner that zinc remains bound to MT as long as high thiol reducing power prevails and is released once the redox balance becomes more oxidizing (7). We here extend these findings by studying the role of glutathione-mediated zinc release in the presence of a zinc acceptor such as an apoenzyme. In particular, we show that only one of the seven zinc atoms i...

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Section: Discussionmentioning

confidence: 97%

The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase

Jiang

Maret

Vallée

1998

Proc. Natl. Acad. Sci. U.S.A.

284

207

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“…Ilcnce, the differences in the actual binding constants explain the present results. As a corollary, the magnitude of the association constant for zinc in ZnT-thionein limits its capacity to transfer the metal to weaker accep-312 furs, Thus, while there are several reports that native metallothionein:t irr vitro reactivat© apometallo~a,~yi=ics with hillher affinity for ~inc [14], it is thermodynamically r,o~ feasible to activate zinc.dependent prot~. "=s of' low affinity with Zn~.th|onein, Differences in bind,rig constant may also explain why in our experiments thionein had no inhibitory effect on the zinc.containing RNA polymerase II in the extract, as illustrated by the unirnpaired transcription with an 'octamer' binding sequence in the promoter (Fig, 2, lane 4), Our in vitro results suggest that changes in the intracellulnr supply of thionein will also affect zinc concentration in rive.…”

Section: Discussionmentioning

confidence: 99%

Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Spl

et al. 1991

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A nuntb¢ r of transcription factors contain so.called zinc finger domains for the inlcr~ction with their cognate DNA sequence, It tins ~¢n shown that rclnOWd or the zinc io,~s complcxed in these zinc t~n~ers abrol~atcs DNA bindin~ and transcription activation, Therefore we wanted to te~ the hypothesis that the activity of tritnscriptton factors could b~ reguhtted by physolo~,ical chel.ttors of zinc, A pron~inent candid~tv,¢ rot such a chdutor is the Cys-rich protci='b thionein (apometallothionein) that is inducible by heavy metal Io=ids, and by oilier environm~nlal stimuli, Here we show with DNA binding, and in Vitro trtmscription ass;¢ys that thionein indeed can in.ctlvat¢ the zinc flnger.containin~ $pl in != reversible manner. By contrast, transcriptio, fitclor Oct-I, which binds DNA via a borneo.domain, i.~, a helix-turn-helix motif not involving zinc ions, is refntctory to thionein action, We propo~ that modulation ofintracellular thlonein concentration is used for the coordinated reguhttion of =l large subset of genes w!mSe tnm~ription depends on zinc finger proteins.

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“…However, the incidence of toxic concentrations of heavy metals in the environment is sporadic; thus, the need to detoxify heavy metals does not seem to account for the ability of virtually all living cells to synthesize metallothioneins transcriptionally or enzymatically (Steffens, 1990;Lazo et al, 1995;Vallee, 1995 Another function proposed for MT is the intracellular regulation of zinc ion levels (Li et al, 1980;Zeng et al, 1991aZeng et al, , 1991bVallee, 1995). MT has been shown to transfer zinc ions to apo-metalloenzymes in vitro, including carbonic anhydrase (Li et al, 1980), aldolase, alkaline phosphatase, themolysin (Udom & Brady, 1980), pyridoxal kinase (Churchich et al, 1989;Hao et al, 1993), and the estrogen receptor (Cano-Gauci & Sarkar, 1996). Phytochelatins (plant metallothioneins) are aiso known to reactivate apo-metailoenzymes (Thumann et al, 1991).…”

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confidence: 99%

Monitoring metal ion flux in reactions of metallothionein and drug‐modified metallothionein by electrospray mass spectrometry

et al. 1998

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The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.Keywords: acquired drug resistance; DNA binding protein; electrospray mass spectrometry; insulin; metal ion flux; metallothionein; stress response Metallothioneins (MT) comprise a family of small metal binding proteins that occurs widely throughout the animal kingdom. Plants and bacteria also produce types of metallothioneins. Vertebrate MTs share essentially the same structure (Kagi, 1993), with 20 cysteines coordinating seven divalent metal cations (Schultze et al., 1988; Robbins et al., 1991). TLVO domains are joined by a linker region, the N-terminal or P-domain binding three metal cations and the C-terminal or cy-domain binding four metal cations. Metallothioneins in invertebrate species also contain multiple cysteine side chains that bind metal cations.Although it would seem that a protein as widespread as MT must play a biological role that is essential for the survival of organisms (Vallee, 1995), the function of MT has been a subject for debate since it was discovered 40 years ago (Margoshes & Vallee, 1957). One function often cited for MT is the detoxification of heavy metals, cadmium in particular. However, the incidence of toxic concentrations of heavy metals in the environment is sporadic; thus, the need to detoxify heavy metals does not seem to account for the ability of virtually all living cells to synthesize metallothioneins transcriptionally or enzymatically (Steffens, 1990;

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Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thionein

Cited by 228 publications

References 43 publications

The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase

The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase

Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Spl

Monitoring metal ion flux in reactions of metallothionein and drug‐modified metallothionein by electrospray mass spectrometry

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