Peptide-Protein Interaction as Studied by Gel Filtration<sup>*</sup>

Fairclough, Gordon F.; Fruton, Joseph S.

doi:10.1021/bi00866a038

Cited by 179 publications

(56 citation statements)

References 30 publications

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“…2). These results are in agreement with findings reported in the literature [20,21] that the indole compounds investigated so far, including glycine peptides of Trp, all bind to HSA at one specific site, and in some cases at a few more very weak sites.…”

Section: Resultssupporting

confidence: 93%

A new approach to the study of the contribution of peptide carriers to antitumour activity; binding of the peptide moiety to human serum albumin

1974

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Section: Resultssupporting

confidence: 93%

A new approach to the study of the contribution of peptide carriers to antitumour activity; binding of the peptide moiety to human serum albumin

1974

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“…Gel filtration experiments [14] were carried out essentially as described by Fairclough and Fruton [15]. A jacketed column (I .25 x 29 cm) of Sephadex G-50 (medium, Pharmacia) was equilibrated at 10 "C with a known concentration of NAD'.…”

Section: Gel Filtration Experimentsmentioning

confidence: 99%

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

et al. 1976

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The binding of oxidized and reduced coenzyme (NAD ' and NADH) to 3-phosphoglyceroylglyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25 '%;) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme . NAD ' complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD' molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 pM at 10 "C, pH 7.0.The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme . NADH complex, since it disappears upon arsenolysis of the acylenzyme.The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD' to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD', the reduced COenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme . NADH complex being estimated as 2.0 pM at 25 "C. In addition a large quenching of the NADH fluorescence (about 83 yo) is observed. The comparison of the dissociation constants of the coenzyme . acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD' . acylenzyme and NADH . acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes.Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.

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“…Solution with P = 0.23, 0.46 and 0.72 (approximately 1, 2 and 3 coenzyme molecules bound to one enzyme molecule) were obtained by gel filtration on Sephadex according to the method of Fairclough and Fruton [23]. The free NAD concentrations corresponding to various degrees of saturation a t 40" and pH 8.…”

Section: Preparation Of Enzyme Solutions At Differentmentioning

confidence: 99%

X‐Ray Small‐Angle Scattering of Yeast Glyceraldehyde‐3‐phosphate Dehydrogenase as a Function of Saturation with Nicotinamide‐Adenine‐Dinucleotide

Durchschlag

Puchwein

Kratky

et al. 1971

European Journal of Biochemistry

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Apo-enzyme revealed a contraction in volume depending upon the degree of saturation.Fully saturated the contraction amounts t o 7O/,. As the observed degree of contraction is not linearly proportional t o the degree of saturation, the simple sequential mechanism cannot explain the observations. To the extent that they coincide with the quantitative predictions of the allosteric model for this particular enzyme, based on calculations involving the equilibrium constants obtained from chemical relaxation experiments under identical conditions, they strongly support the latter mechanism.Comparison of the experimental scattering curves with theoretical scattering curves of compact full cylinders did not bring about satisfactory agreement in the range of the fist submaximum.Hollow circular cylinders with h: 2r, = 0.6 : 1 (apo-enzyme) or h : 2r1 = 0.58 : 1 (holo-enzyme) may be regarded as sufficiently equivalent in scattering as far as the form of the main maximum is concerned as well as the position and relative intensity of the submaximum. Even if the molecule consists of four subunits they consequently must be arranged in such a way that the entire particle is very similar to a hollow cylinder. The nature of the structural changes occurring in the allosteric transition can now be described in terms of an increase of the inherent anisotropy and of a considerable decrease in the solvent included in the tetrameric assembly.Exposure of the enzyme to 40" over a longer period of time leads t o sigdcant alterations in the scattering curves. These can be interpreted on the basis of growing aggregates in polydisperse solutions. This process is accompanied by a considerable decrease of enzyme activity.

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Peptide-Protein Interaction as Studied by Gel Filtration^*

Abstract: The results presented in this communication will form part of the dissertation of Gordon F. Fairclough, Jr., in partial fulfillment of the requirements for the Ph.D. degree from Yale University.

Cited by 179 publications

References 30 publications

A new approach to the study of the contribution of peptide carriers to antitumour activity; binding of the peptide moiety to human serum albumin

A new approach to the study of the contribution of peptide carriers to antitumour activity; binding of the peptide moiety to human serum albumin

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

X‐Ray Small‐Angle Scattering of Yeast Glyceraldehyde‐3‐phosphate Dehydrogenase as a Function of Saturation with Nicotinamide‐Adenine‐Dinucleotide

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Peptide-Protein Interaction as Studied by Gel Filtration*

Abstract: The results presented in this communication will form part of the dissertation of Gordon F. Fairclough, Jr., in partial fulfillment of the requirements for the Ph.D. degree from Yale University.

Cited by 179 publications

References 30 publications

A new approach to the study of the contribution of peptide carriers to antitumour activity; binding of the peptide moiety to human serum albumin

A new approach to the study of the contribution of peptide carriers to antitumour activity; binding of the peptide moiety to human serum albumin

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

X‐Ray Small‐Angle Scattering of Yeast Glyceraldehyde‐3‐phosphate Dehydrogenase as a Function of Saturation with Nicotinamide‐Adenine‐Dinucleotide

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Peptide-Protein Interaction as Studied by Gel Filtration^*