The reactivity and function of cysteine residues in rabbit liver aldolase B

Heyduk, Tomasz; Moniewska, A.; Kochman, Marian

doi:10.1016/0167-4838(86)90033-6

Cited by 10 publications

(6 citation statements)

References 38 publications

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“…The modified protein was purified on a Sephadex G-25 column equilibrated with Bis-Tris buffer at pH 6.0, a pH which inhibits -SH ^-SSinterchange reactions (Parker & Kharasch, 1959). Then the amount of TNB per mole of CRP was determined spectrophotometrically as described in Heyduk et al (1986) or by measuring the amount of TNB released by DTT treatment of the modified protein. In both cases, 1.9 TNB/mol of CRP was obtained, and, therefore, no intersubunit disulfide bridges were formed in this case.…”

Section: Resultsmentioning

confidence: 99%

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Heyduk¹,

Lee²

1989

Biochemistry

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136

232

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Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.

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Section: Resultsmentioning

confidence: 99%

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Heyduk¹,

Lee²

1989

Biochemistry

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136

232

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“…One was bovine pancreas chymotrypsinogen (Worthington, Freehold, NJ), which has five disulfide linkages (37). The second was rabbit muscle aldolase (Worthington), which has eight free sulfhydryl groups/subunit but no disulfide linkages (38).…”

Section: Methodsmentioning

confidence: 99%

Pyrrolidine Dithiocarbamate Prevents p53 Activation and Promotes p53 Cysteine Residue Oxidation

Momand

1998

Journal of Biological Chemistry

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Pyrrolidine dithiocarbamate (PDTC) is a thiol compound widely used to study the activation of redox-sensitive transcription factors. Although normally used as an antioxidant, PDTC has been shown to exert pro-oxidant activity on proteins both in vitro and in vivo. Because p53 redox status has been shown to alter its DNA binding capability, we decided to test the effect of PDTC on p53 activation. In this communication, we report that PDTC inhibits the activation of temperature-sensitive murine p53Val-135 (TSp53) in the transformed rat embryo fibroblast line, A1-5, as well as wild-type human p53 in the normal diploid fibroblast line, WS1neo. In A1-5 cells, PDTC abrogated UV-and temperature shift-induced TSp53 nuclear translocation and p53-mediated transactivation of MDM2. PDTC also blocked UV-induced accumulation of wild-type p53 in WS1neo cells. Continual presence of PDTC was required for its effect as both UV-induced nuclear translocation and accumulation resumed after PDTC removal. We next investigated whether PDTC treatment altered the p53 redox state. We found that PDTC increased p53 cysteine residue oxidation in vivo. This represents the first direct evidence showing that the p53 redox state can be altered in vivo and that increased oxidation correlates with its inability to perform its downstream functions.

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“…While these cysteine residues are not directly involved in the catalytic mechanism, cysteine conjugation inhibits enzyme activity, possibly through the prevention of conformational changes required for catalysis (6,7). The fast-reacting thiols of rabbit aldolase, also not directly involved in catalysis (8), only react with a second order rate constant of 13.0 to 2.62 ϫ 10 2 M Ϫ1 s Ϫ1 (9,10). Rat hemoglobin has been shown to possess a cysteine residue, Cys-125␤, whose reaction with DTNB has a rate constant of 2.94 ϫ 10 4 M Ϫ1 s Ϫ1 (11).…”

mentioning

confidence: 98%

Highly Reactive Cysteine Residues in Rodent Hemoglobins

Miranda

2000

Biochemical and Biophysical Research Communications

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The reactivity and function of cysteine residues in rabbit liver aldolase B

Cited by 10 publications

References 38 publications

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Pyrrolidine Dithiocarbamate Prevents p53 Activation and Promotes p53 Cysteine Residue Oxidation

Highly Reactive Cysteine Residues in Rodent Hemoglobins

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