Bovine neurophysin I dimerization studied by rapid kinetic techniques

Af, Pearlmutter

doi:10.1021/bi00576a006

Cited by 15 publications

(36 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…12B) is compatible with the similar rate constants found for dimerization of the unliganded protein (56) and for peptide binding to monomer (145). Both these values are -lo5 M-' s -' (56,145) as opposed to -lo6 M-' s -' for peptide binding to dimer (63,64).…”

Section: Recognition and Function In Neurophysin-hormone Systems 55supporting

confidence: 70%

“…(46, SO)] and by marked similarities in most other properties. The single clear exception to this generality is the reaction of bovine neurophysin-I (oxytocin associated) with bromophenol blue, which is not paralleled by the vasopressin-associated bovine neurophysin-I1 (56,57). Rat neurophysins also appear to differ among themselves in reactivity towards bromophenol blue (58), but it has not been established whether this apparent difference between classes of neurophysins can be generalized.…”

Section: A Comparative Neurophysin Propertiesmentioning

confidence: 99%

See 1 more Smart Citation

Molecular, Thermodynamic, and Biological Aspects of Recognition and Function in Neurophysin‐Hormone Systems: A Model System for the Analysis of Protein–Peptide Interactions

Breslow

Burman

1990

Advances in Enzymology - And Related Areas of Molecular Biology

View full text Add to dashboard Cite

Section: Recognition and Function In Neurophysin-hormone Systems 55supporting

confidence: 70%

Section: A Comparative Neurophysin Propertiesmentioning

confidence: 99%

Molecular, Thermodynamic, and Biological Aspects of Recognition and Function in Neurophysin‐Hormone Systems: A Model System for the Analysis of Protein–Peptide Interactions

Breslow

Burman

1990

Advances in Enzymology - And Related Areas of Molecular Biology

View full text Add to dashboard Cite

“…The latter may occur between the strong and weak sites on a single polypeptide X 10-P M) was equilibrated with dye by gel filtration or equilibrium (initial concentration 9.5 X 10-f M) was equilibrated with dye by gel chain or, more likely, between two strong sites on a dimer; as described later, bound dye is located predominantly on the dimer. The strong site we observe at pH 4 appears (from the concentration conditions used) to be the principal site studied by Pearlmutter (1979) since her kinetic studies were conducted at low ratios of dye to protein. Data at the lowest values of 3 in fact give an apparent binding constant virtually identical with that obtained by Pearlmutter (1979).…”

Section: Resultsmentioning

confidence: 87%

“…The highest values of P shown in each plot represent the highest values attainable, under the conditions used, that were not accompanied by precipitation of the protein-dye complex. The pH 4 data are of particular interest because this pH was the least complicated by precipitation and was the pH at which thermodynamic measurements were made by Pearlmutter (1979). These results indicate the presence of a single strong site and one (or more) weak sites per polypeptide chain.…”

Section: Resultsmentioning

confidence: 99%

Interaction of bromophenol blue and related dyes with bovine neurophysin-I: use as a probe of neurophysin chemistry

Carlson¹,

Breslow²

1981

Biochemistry

View full text Add to dashboard Cite

The interaction of bromophenol blue and related dyes with bovine neurophysin-I was studied by equilibrium dialysis and gel filtration, absorption and circular dichroism spectroscopy, and analytical ultracentrifugation. Binding isotherms for bromophenol blue showed positive cooperativity, with one strong site and one or more weaker sites present per polypeptide chain at pH 4 and an apparent increase in relative importance of the weaker sites of lower pH. Circular dichroism (CD) studies suggested displacement of bound dye by peptides that bind to the neurophysin hormone binding site. Titration of bound bromophenol blue indicated that the deprotonated dye was bound to the strong site with approximately 20-fold greater affinity than the protonated dye. The pH dependence of binding of bromophenol blue and of bromocresol purple, which has a higher pKa than bromophenol blue, indicated that binding was dependent on protonation of a protein residue with a pKa of 2.9. This residue was identified as a protein carboxyl, probably on an abnormal side chain, by studies of glycine ethyl ester modified neurophysin and carboxypeptidase-treated neurophysin. The presence of exciton interactions between bound dye molecules when only one dye was bound per polypeptide chain and analytical ultracentrifugation results indicated that dye was bound predominantly to the dimeric form of the protein. The implication of the data are discussed with respect to a kinetic model of dye-neurophysin interaction, used elsewhere in a study of neurophysin dimerization, that assumed interaction of protein monomers with protonated dye. Additionally, results are presented which suggest, in disagreement with conclusions based on the kinetic model, that there is a pH-dependent component of neurophysin dimerization which parallels low pH fluorescence and CD changes observed earlier.

show abstract

“…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). "slue for dissociation of BNP I dimer (Pearlmutter, 1979); the k.., value for oxytocin binding to BNP I1 monomer, a value also relevant to compare, is 2 s-I (Pearlmutter & Dalton, 1980b). pendent upon the concentration of soluble protein ( Figure 6).…”

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

confidence: 99%

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

Swaisgood¹,

Chaiken²

1986

Biochemistry

View full text Add to dashboard Cite

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)

show abstract

Bovine neurophysin I dimerization studied by rapid kinetic techniques

Cited by 15 publications

References 17 publications

Molecular, Thermodynamic, and Biological Aspects of Recognition and Function in Neurophysin‐Hormone Systems: A Model System for the Analysis of Protein–Peptide Interactions

Molecular, Thermodynamic, and Biological Aspects of Recognition and Function in Neurophysin‐Hormone Systems: A Model System for the Analysis of Protein–Peptide Interactions

Interaction of bromophenol blue and related dyes with bovine neurophysin-I: use as a probe of neurophysin chemistry

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

Contact Info

Product

Resources

About