Analysis of self-association of bovine neurophysins by gel chromatography

Whittaker, Barbara A.; Allewell, Norma M.

doi:10.1016/0003-9861(84)90308-4

Cited by 13 publications

(11 citation statements)

References 22 publications

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“…The results indicate a single transition with an apparent midpoint, K¿, at 2 X 10"4 M, giving a dimerization constant of 5 X 103 M'1. This value compares well with dimerization constants of (7-11) X 103 M'1 calculated for native and nitrated neurophysin I at pH 5.6 (Nicolas et al, 1978(Nicolas et al, , 1980; Whittaker & Allewell, 1984), particularly when the increase in dimerization constant of neurophysin I with decreasing pH (Peyton et al, 1986) is considered.…”

Section: Resultssupporting

confidence: 79%

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

Breslow¹,

Laborde²,

Bamezai³

et al. 1991

Biochemistry

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The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.

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Section: Resultssupporting

confidence: 79%

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

Breslow¹,

Laborde²,

Bamezai³

et al. 1991

Biochemistry

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“…The dimerization constant was evaluated by means of Eqn (1 1) from a plot of 1/( V -Vo) as a function of the concentration of neurophysin applied to the initial zone, since the total protein concentration in the zone is unknown due to spreading. The close agreement with literature values for the soluble protein (Whittaker and Allewell, 1984) shows that immobilization on the high performance affinity supports described above does not alter the self-association properties of neurophysin, at least statistically. In contrast, with Sepharose-bound neurophysin, a dimerization constant that is roughly 10-fold higher than in solution was obtained (Angal and Chaiken, 1982;Chaiken et a/., 1983).…”

Section: Neuroph Ysinsupporting

confidence: 89%

Interaction between immobilized and soluble protein subunits. Analysis and applications

Chiancone

Gattoni

1990

J of Molecular Recognition

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A distinctive property of oligomeric and self-associating proteins is the high specificity of the subunit recognition process. Protein subunits immobilized covalently on a solid matrix maintain this characteristic and are able to bind soluble subunits of the same or a closely related protein under conditions that allow the establishment of a finite association/dissociation equilibrium. The basic theory for studying the immobilized-soluble subunit interaction is presented together with the methodology for a proper protein immobilization. Specific examples are discussed to illustrate on the one hand benefits and caveats of using immobilized protein subunits to measure interaction constants, and on the other preparative applications of subunit affinity columns.

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“…The use of immobilized BNP I1 limits the binding process measured to that of peptide ligand largely to immobilized protein mon~m e r .~ The concentration of [3H]AVP was varied over a sufficiently wide range that the concentration dependence predicted theoretically (eq 13) was observed on the NPG matrix ( Figure 4). In contrast, no clearcut concentration dependence was observed with the CPG matrix (Figure 8 On the basis of the dimer association constant reported by Whittaker and Allewell (1984) of 1.1 X lo4 M-' for BNP I1 in 0.1 M phosphate buffer, pH 5.6, and the expected similarity of (or small decrease in) such values at pH 7 (Whittaker & Allewell, 1984;Tellam & Winzor, 1980;Nicolas et al, 1980), 74% and 72% of the BNP I1 molecules would be expected to be monomers in the immobilization solutions for CPG and NPG, respectively. Given the relatively low concentrations of dimers in the reaction solutions and the fact that both monomeric partners of the dimer would have to be covalently attached for protein to remain immobilized as dimers after washing, the probability is low for dimer immobilization.…”

Section: Discussioncontrasting

confidence: 45%

“…e Values obtained: 16-20 p M for binding of oxytocin to soluble BNP I1 from equilibrium dialysis studies (Nicolas et al, 1978;Tellam & Winzor, 1980); 50 pM for binding of hormone to soluble BNP I1 from competitive binding quantitative affinity chromatography (Angal & Chaiken, 1982); and 6.7 pM for binding of [Lys8]vasopressin to soluble BNP I1 dimer (Pearlmutter & Dalton, 1980a). /Values obtained for dimerization of unligand monomer under conditions similar to those used here: by analytical gel chromatography, 56-91 p M (Whittaker & Allewell, 1984; and by sedimentation equilibrium, 118-172 p M (Tellam & Winzor, 1980;Nicolas et al, 1976Nicolas et al, , 1980. Walue for dissocia.tion of [Lys*]vasopressin from singly liganded BNP I1 dimer (Pearlmutter & Dalton, 1980a); the value for [Lys8]vasopressin from doubly liganded BNP I1 dimer is 6 s-I (Pearlmutter & Dalton, 1980a).…”

Section: Chromatographic Evaluation Of Equilibrium Constants For [3h]mentioning

confidence: 96%

Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrixes

Swaisgood¹,

Chaiken²

1986

Biochemistry

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Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)

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Analysis of self-association of bovine neurophysins by gel chromatography

Cited by 13 publications

References 22 publications

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

Interaction between immobilized and soluble protein subunits. Analysis and applications

Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrixes

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