Measurement of protein-binding phenomena by gel filtration

Hummel, John P.; Dreyer, William J.

doi:10.1016/0006-3002(62)90124-5

Cited by 1,280 publications

(499 citation statements)

References 5 publications

Supporting

Mentioning

486

Contrasting

Order By: Relevance

“…This module might be regulated by nucleotides, because CVAK104 is an ATP-binding protein (Conner and Schmid, 2005). Using the gel chromatographic procedure of Hummel and Dreyer (1962), we also observed ATP binding to CVAK104 (our unpublished data). Sequence comparisons between species ranging from mammals to fish indicate that CVAK104 is a highly conserved protein.…”

Section: Discussionmentioning

confidence: 79%

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

Düwel

Ungewickell

2006

MBoC

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 79%

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

Düwel

Ungewickell

2006

MBoC

View full text Add to dashboard Cite

show abstract

“…The binding of a large receptor to a small ligand can be studied by gel filtration catTied out at equilibrium, that is, in the presence of ligand (24). As the receptor emerges from the column, a peak and then a trough appear in the concentration of the ligand (Fig.…”

Section: Equilibrium Gel Filtrationmentioning

confidence: 99%

“…Attie and Raines Page 14 Elution profile of the absorbance at 285 nm accompanying the passage of the protein ribonuclease A through a gel-filtration column, which was equilibrated with the ligand 2′-cytidylic acid (24).…”

Section: Activated Charcoalmentioning

confidence: 99%

Analysis of Receptor-Ligand Interactions

Attie¹,

Raines²

1995

J. Chem. Educ.

View full text Add to dashboard Cite

Molecules have an innate affinity for one another due to electrostatic forces, such as Coulombic attractions, hydrogen bonds, and dispersion forces. The noncovalent interactions that result from this affinity are of particular importance in biological processes, including the catalysis of chemical reactions (by enzymes), neutralization of foreign toxins (by antibodies), and stimulation of cellular activities (by hormones). To initiate these processes, receptors and ligands exchange interactions with solvent and solute molecules for interactions with each other. Much effort is currently being made by biological chemists to understand the molecular details of receptor-ligand interactions, and by medicinal chemis ts to exploit this understanding in developing useful pharmaceutics (1). In addition, organic chemists are attempting to develop s synthetic systems that mimic the biological interactions (2, 3). Each of these efforts requires knowledge of the number of potential binding sites on a receptor and the affinity of each binding site for its ligand. In this article, we describe how this knowledge can be obtained, and draw parallels, when appropriate, to the analysis of the kinetics of enzymatic catalysis (4, 5). Properties of Receptor-Ligand InteractionsThe interactions of all biological receptors with their natural ligands share several properties. SpecificityBiological receptors generally bind tightly to a single natural ligand. (This specificity need not be absolute. For example, a number of natural toxins, such as α-bungarotoxin, carry out their mischief by binding to the acetylcholine receptor.) Ligand specificity can be readily assessed through a competitive-binding assay. Here, the amount of ligand bound to a receptor is measured in the presence of other putative ligands. If the receptor is indeed specific for the original ligand, the amount of ligand bound is not affected by the presence of the other ligands. For example, the addition of a 1000-fold molar excess of serum albumin does not decrease the amount of diphtheria toxin bound to its cell-surface receptor. AffinityMolecules interact noncovalently with other molecules. For example, proteins tend to stick to glass, due in part to polar surfaces interacting with one another. The surface of a cell is also quite polar, due largely to the extensive amount of carbohydrate that extends from membrane proteins and membrane lipids. Consequently, all proteins have some affinity for cell surfaces. Receptor-ligand interactions are distinguished from other noncovalent interactions between molecules by their high affinity.

show abstract

“…Nucleotide Binding-Hummell and Dreyer equilibrium gel filtration experiments were used to measure ATP and ADP binding by ParA (15,16). Binding was determined as the amount of ATP and/or ADP that co-eluted with ParA through a gel filtration column.…”

Section: Methodsmentioning

confidence: 99%

“…We used equilibrium gel filtration (15,16), which measures ATP binding as the amount of ATP that co-elutes with protein over a gel filtration column. Since this is an equilibrium technique, one can deduce ATP stoichiometry as well as binding affinity.…”

Section: Atp and Adp Binding Bymentioning

confidence: 99%

Modulation of the P1 Plasmid Partition Protein ParA by ATP, ADP, and P1 ParB

Davey

Funnell

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

ParA is an essential P1 plasmid partition protein. It represses transcription of the par genes (parA and parB) and is also required for a second, as yet undefined step in partition. ParA is a ParB-stimulated ATPase that binds to a specific DNA site in the par promoter region. ParA is an essential component of the P1 plasmid partition system (1). The prophage of bacteriophage P1 exists as an autonomously replicating plasmid, and its copy number is about the same as that of the bacterial chromosome (2). The P1 partition system, Par, directs proper segregation and thus stable maintenance of the plasmid. The mechanism of partition is unknown, but can be thought of as a positioning process that via interaction with the Escherichia coli host ensures proper distribution of the plasmid. Par encodes two essential transacting proteins, ParA and ParB, and contains a cis-acting DNA site called parS (1). In addition, the E. coli integration host factor, IHF, 1 also participates, although it is not absolutely required (3). ParB and IHF bind to parS to form the partition complex (3-5). Assembly of this complex is assumed to be an early step in the partition pathway. Formation of this complex does not require the action of ParA (3-5), and we infer that ParA acts during a later step in the partition process.ParA has at least two roles in partition. First, ParA represses transcription of its own gene and parB from a promoter upstream of parA (6). ParA repressor activity is stimulated by ParB; however, ParB has no effect on par gene expression on its own. A second role for ParA in partition is inferred from genetic data that show a requirement for ParA in partition even when its regulatory role is bypassed (6, 7). Therefore, we consider ParA's repressor activity its "regulatory" function, and this second, as yet undefined role as ParA's "partition" function because we think it reflects a direct role for ParA in the positioning reaction. The latter function requires that ParA interact, directly or indirectly, with the ParB-IHF-parS partition complex.ParA is an ATPase and a site-specific DNA-binding protein (8). The ATPase is stimulated by ParB and nonspecific DNA (8). ParA is one of the better characterized proteins in a superfamily of ATPases defined by a modified Walker type A motif in the protein sequence (9 -11). This superfamily includes other plasmid partition proteins such as F SopA, as well as plasmid and chromosomally encoded proteins from various bacteria species whose functions have not yet been determined (10 -12). Many of the plasmids and bacterial chromosomes also encode a ParB homolog adjacent to the ParA homolog. The similarities of these ParA-like proteins with P1 ParA and F SopA have led to the suggestion that these homologs are also involved in plasmid or chromosome segregation (10, 11).ParA binds to the par promoter region called parOP (8), and this binding is thought to mediate ParA repressor activity in vivo. The recognition site for ParA is likely a large inverted repeat in the par promoter region, since (i) ...

show abstract

Measurement of protein-binding phenomena by gel filtration

Cited by 1,280 publications

References 5 publications

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

Analysis of Receptor-Ligand Interactions

Modulation of the P1 Plasmid Partition Protein ParA by ATP, ADP, and P1 ParB

Contact Info

Product

Resources

About