On the reactivity of metallothioneins with 5,5′-dithiobis-(2-nitrobenzoic acid)

Li, T Y; Minkel, Daniel T.; Shaw, C. Frank; Petering, David H.

doi:10.1042/bj1930441

Cited by 75 publications

(49 citation statements)

References 15 publications

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“…We have suggested that this free thiol might be more reactive toward disulfides such as GSSG (4). Herein, we have used Ellman's reagent to investigate the redox behavior of MT, a reaction that has been studied earlier in some detail (13,14,34). Under the conditions of our experiments, the kinetic phase affected by ATP corresponds to the faster of the two phases ascribed by others to the reaction of the ␣-domain of MT (35).…”

Section: Discussionmentioning

confidence: 99%

The ATP–metallothionein complex

Jiang

Maret

Vallée

1998

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

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We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (K d ‫؍‬ 176 ؎ 33 M, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol-disulfide interchange with Ellman's reagent [5,5-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di-or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two critical cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding constant and the cellular concentration of ATP indicate that in the cell MT could be essentially saturated with ATP at low concentrations of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.Seven zinc atoms bound to 20 cysteine sulfur atoms form the two metal clusters of metallothionein (MT) and constitute a reservoir for storage and distribution of zinc (1). The cluster structure forms a network of zinc-sulfur interactions that have no precedent in the nonliving world. The protein envelops the zinc atoms, each of which is bound to four thiolate ligands, in a manner that effectively shields them from the environment. Ligand exchange and redox reactions of the cysteine sulfur donor atoms are responsible for the kinetic lability of zinc in the thermodynamically stable sites of MT (2). The cluster unit works by a mechanism that allows the cysteine sulfur ligands to zinc to be oxidized, allowing an oxidoreductive mechanism to modulate affinity of the otherwise inert zinc atom (3). We have shown further that the interactions of MT with glutathione (GSH) and glutathione disulfide (GSSG) (4) or other oxidizing agents (5) control the state of zinc in MT in vitro, suggesting that the zinc content of MT is a function of the cellular redox state. Aside from GSH (6), the only other biological ligand known to bind to MT, until now, is phosphate that appears to be required for the formation of stable MT crystals (7). It was found to be localized between the two protein domains bound to the carbonyl group of Cys-19 and to the -amino group of Lys-31, which forms the only interdomain hydrogen bonds with the carbonyl groups of Cys-19 and Cys-21 (7). It has also been found to bind to dimeric Cd-MT with relativ...

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Section: Discussionmentioning

confidence: 99%

The ATP–metallothionein complex

Jiang

Maret

Vallée

1998

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

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“…The assay for free sulfhydryl groups used 5,5Ј-dithiobis(2-nitrobenzoic acid) as described in ref. 37.…”

Section: Preparation Of T and T(o) And Assay Of T(o)mentioning

confidence: 99%

Thionein can serve as a reducing agent for the methionine sulfoxide reductases

Sagher

Brunell

Hejtmancik

et al. 2006

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

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“…The initial reaction is characteristic of free thiol reacting with 5,5'-dithiobis-(2-nitrobenzoic acid), according to studies with horse-kidney 01 metallothionein (Li et al, 1979). The second phase is similar to the reaction observed between 5,5'-dithiobis-(2-nitrobenzoic acid) and thiol groups bound to cadmium, zinc, or copper in the horse kidney protein (Li et al, 1979 . In tumours from zinc-deficient animals, not only was the incorporation of thymidine into DNA greatly depressed, but its uptake into the cells was also greatly inhibited.…”

Section: Characterization Of the Cadmium-and Zinc-binding Proteinmentioning

confidence: 58%

“…As is described elsewhere, colour development is time-dependent, and the increase in absorbance at 412 nm must be followed until the reaction is complete in order to obtain valid results (Li et al, 1979).…”

Section: Preparation Ofcellfractionsmentioning

confidence: 99%

Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells

Koch¹,

Wielgus²,

Shankara³

et al. 1980

Biochemical Journal

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The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc-and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc-and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.

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On the reactivity of metallothioneins with 5,5′-dithiobis-(2-nitrobenzoic acid)

Cited by 75 publications

References 15 publications

The ATP–metallothionein complex

The ATP–metallothionein complex

Thionein can serve as a reducing agent for the methionine sulfoxide reductases

Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells

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