The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase

Hunt, John B.; Neece, Sue H.; Ginsburg, Ann

doi:10.1016/0003-2697(85)90409-9

Cited by 226 publications

(268 citation statements)

References 26 publications

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“…Zinc Measurements-The zinc content of DnaJ was determined using a combined PAR/PMPS assay (23). In this assay, free zinc readily interacts with 4-(2-pyridylazo) resorcinol (PAR) (Sigma) to form a Zn-(PAR) 2 complex, which strongly absorbs at 500 nm.…”

Section: Construction Of Zinc Center Mutants Of Dnaj-mentioning

confidence: 99%

“…To determine whether the zinc centers can form independently of each other, the zinc content of the two mutant proteins and wild-type DnaJ was analyzed using the PAR/PMPS titration assay (23,24). In this assay, the total amount of zinc, which is specifically coordinated via thiol groups in a protein, can be determined.…”

Section: Zinc Center II Essential For In Vivo Function Of Dnaj-thementioning

confidence: 99%

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The Roles of the Two Zinc Binding Sites in DnaJ

Linke

Wolfram

Bussemer

et al. 2003

Journal of Biological Chemistry

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All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/ GrpE foldase machinery catalytically active.

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Section: Construction Of Zinc Center Mutants Of Dnaj-mentioning

confidence: 99%

Section: Zinc Center II Essential For In Vivo Function Of Dnaj-thementioning

confidence: 99%

The Roles of the Two Zinc Binding Sites in DnaJ

Linke

Wolfram

Bussemer

et al. 2003

Journal of Biological Chemistry

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“…It has been shown that zinc-DTT complexes can be formed (Cornell and Crivaro, 1972;Hunt et al, 1985) but DTT at concentrations up to 40 mM induced no loss of the intrinsic zinc in p53 even during denaturation of the protein (Bullock et al, 1997). On the other hand, it can be anticipated that DTT will decrease the concentration of free Zn 2+ ions.…”

Section: Micromolar Concentrations Of Zn 2+ Ions Inhibit Binding Of Pmentioning

confidence: 99%

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments

et al. 1999

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Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNAp53 complex. The intensity and the number of these bands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 mM zinc ions do not in¯uence p53 binding to p53CON in a DNA oligonucleotide, we show that 5 ± 20 mM zinc e ciently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory e ect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less e ciently than zinc; cobalt ions are least e cient, requiring 4100 mM Co 2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.

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“…Determination of sulphydryl groups. Sulphydryl groups were determined according to the method of Habeeb (15), by p-hydroxymercuriphenylsulfonic acid (PMPS) titration (16) and by polyacrylamide gel electrophoresis (17). The method of Habeeb was performed with some modifications as follows: 23~g of pure CDA were dialyzed extensively against 20mM Tris/HC1 buffer pH 7.5 to remove DTT.…”

Section: Methodsmentioning

confidence: 99%

Human placenta cytidine deaminase: a zinc metalloprotein

et al. 1997

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Summary: Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10-phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolirtic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents.

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The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase

Cited by 226 publications

References 26 publications

The Roles of the Two Zinc Binding Sites in DnaJ

The Roles of the Two Zinc Binding Sites in DnaJ

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments

Human placenta cytidine deaminase: a zinc metalloprotein

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