Quantitative studies of rapid polymerization equilibrium by gel filtration. The z-average elution volume of a reaction boundary

Winzor, Donald J.; Loke, Joan P.; Nichol, L.W.

doi:10.1021/j100872a053

Cited by 18 publications

(5 citation statements)

References 8 publications

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“…Consequently, the data conform very well with the law of mass action, written in logarithmic form, for a monomer-dimer system. This result thus confirms earlier claims (Winzor & Scheraga, 1964;Winzor et al, 1967) that a-chymotrypsin undergoes reversible dimerization under these conditions. Furthermore, the mean value of 3.5 ± 0.1 L/g for the dimerization constant that is deduced from all of the [aA(r), c(r)] data is in very good agreement with previous estimates of 3.8 (Winzor & Scheraga, 1964), 7 (Winzor et al, 1967), 1: Analysis of equilibrium sedimentation experiments on -chymotrypsin in I = 0.2 acetate-chloride, pH 3.86, by means of the ( ) analysis (Milthorpe et al, 1975): representative plot of the extrapolation involved in determining aA(rF), the activity of monomer at reference total enzyme concentration c(rF), which has been fixed at 1.87 g/L in each of five experiments.…”

Section: Resultssupporting

confidence: 92%

“…Because of the uncertainty created by these conflicting viewpoints, we have investigated the binding of /V-acetyl-Ltryptophan to -chymotrypsin in / = 0.2 acetate-chloride, pH 4, a medium in which (a) polymerization of the enzyme is restricted to a monomer-dimer equilibrium (Winzor & Scheraga, 1964; Winzor et al, 1967; Morimoto & Kegeles, 1967; Auné & Timasheff, 1971; Horbett & Teller, 1973Gorbunoff et al, 1978) and (b) the problem of autolysis in equilibrium binding studies by frontal gel chromatography (Nichol et al, 1972) is minimal. A combination of equilibrium sedimentation, gel chromatographic, and enzyme kinetic studies has been used to evaluate the binding constants for the interactions of /V-acetyl-L-tryptophan with monomeric and dimeric a-chymotrypsin.…”

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confidence: 99%

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Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of .alpha.-chymotrypsin

Tellam¹,

Jersey²,

Winzor³

1979

Biochemistry

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The binding of N-acetyl-tryptophan to the monomeric and dimeric forms of alpha-chymotrypsin in I = 0.2 acetate-chloride buffer, pH 3.86, has been studied quantitatively. Equilibrium sedimentation studies in the absence of inhibitor yielded a dimerization constant of 3.5 L/g. This value was confirmed by frontal gel chromatography of the enzyme on Bio-Gel P-30, which was also used to establish that N-acetyl-L-tryptophan binds preferentially to monomeric enzyme. From kinetic studies of competitive inhibition with N-acetyl-L-tryptophan ethyl ester as substrate, an equilibrium constant of 1300 M-1 was determined for the binding of N-acetyl-L-tryptophan to monomeric alpha-chymotrypsin. An intrinsic binding constant of 250 M-1 for the corresponding interaction with dimeric enzyme was calculated on the basis of these results and binding data obtained with concentrated (18.5 g/L) alpha-chymotrypsin. The present results refute earlier claims for exclusive binding of competitive inhibitors to monomer and also those for equivalence of inhibitor binding to monomeric and dimeric forms of alpha-chymotrypsin.

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Section: Resultssupporting

confidence: 92%

mentioning

confidence: 99%

Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of .alpha.-chymotrypsin

Tellam¹,

Jersey²,

Winzor³

1979

Biochemistry

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“…The stability of the CheA dimer was investigated by frontal gel filtration at 4°C (22). No change in the elution profile was observed for CheA over a 15-fold change in concentration ranging from 3 ,uM to 45 ,uM (data not shown).…”

Section: Resultsmentioning

confidence: 96%

Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA.

Gegner

Dahlquist

1991

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

187

148

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An essential step in the signal transduction pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCI/5 mM MgCl2, pH 7.4 at 40C) was determined to be in the physiologically relevant range of 17 FAM. No evidence for cooperativity in the association of CheW with CheA was found. CheW also plays a role in coordinating the kinase activity of CheA in the adaptation response. The cell adapts to stimuli by adjusting the methylation state of its receptors (9). Adaptation to attractant results in an increase in the methylation state ofthe receptor, whereas adaptation to a repellent results in a decrease. In the repellent adaptation response, CheA phosphorylates CheB (the methylesterase), which is then activated to demethylate the receptor and restore the cell to its prestimulus swimming state of random tumbles and smooth swims (10, 11). Demethylation in response to repellent requires both CheA and CheW. During the adaptation response to attractant stimuli, the methylesterase is transiently inhibited. The mechanism responsible for methylesterase inhibition is not fully understood, but the adaptation response to positive stimuli appears to require only CheW (11). This observation suggests that parts of a CheWdependent signaling pathway may be independent of CheA.We have initiated studies of the physical interactions between CheW and the other components of the signal transduction and adaptation pathways of the chemotaxis system. Through such experiments, we hope to obtain a more detailed picture of the molecular pathway involved in chemotaxis signal transduction. It is not known how the kinase activity of CheA is coupled to the receptor by CheW, but the lack of any known CheW covalent modification suggests that complex formation between CheW and CheA may be involved. Here we report the results of our investigation that demonstrate an interaction between monomeric Mr 18,000 CheW and dimeric Mr 142,000 CheA. The ultimate goal ofthis work is to understand the molecular events that control swimming behavior in response to chemotactic stimuli. It appears that the formation of reversible complexes among various chemotaxis proteins may be an important feature of these molecular events. MATERIALS AND METHODSBacterial Strains and Plasmids. The cheW overexpression plas...

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“…The association equilibrium was quantified in terms of stoichiometry ( n ) and association constant ( K ). Assignment of magnitudes to the elution volumes of monomer for two-state self-association ( n M ⇆ P) allowed the weight-fraction of monomer, f M , in each applied solution to be calculated as

f_{M} = (V_{w} - V_{P}) ∕ (V_{M} - V_{P})

The reaction stoichiometry ( n ) was deduced [24] by analyzing the [ C t , f M ] data set in terms of the logarithmic transform of the law of mass action for two-state self-association ( n M ⇆ P) governed by association constant K , namely

log [(1 - f_{M}) C_{t}] = log (n K) + n log (f_{M} C_{t})

where C t is the base-molar concentration (weight-concentration divided by monomer molecular mass). To avoid the bias of statistical weighting imposed by use of the linear transform, K was obtained by direct analysis of the [ C t , f M ] data set in terms of the untransformed relationship

(1 - f_{M}) C_{t} = n K {(f_{M} C_{t})}^{n}

using the integer value of n deduced from the slope of the linear dependence of log [(1 – f M ) C t ] upon log ( f M C t ).…”

Section: Discussionmentioning

confidence: 99%

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

Pazehoski

Cobine

Winzor

et al. 2011

Biochemical and Biophysical Research Communications

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Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin binding domain of Streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

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Quantitative studies of rapid polymerization equilibrium by gel filtration. The z-average elution volume of a reaction boundary

Cited by 18 publications

References 8 publications

Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of .alpha.-chymotrypsin

Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of .alpha.-chymotrypsin

Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA.

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

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