The Preparation and Some Properties of Fibrinogen Precipitated from Human Plasma by Glycine.

Kazal, Louis A.; Amsel, Sheldon; Miller, Orin P.; Tocantins, Leandro M.

doi:10.3181/00379727-113-28553

Cited by 233 publications

(112 citation statements)

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“…Ligands-The ligands fibronectin (24), vitronectin (25), and fibrinogen (26) were purified from human blood and biotinylated as described previously (27,28).…”

Section: Methodsmentioning

confidence: 99%

Definition of an Unexpected Ligand Recognition Motif for αvβ6 Integrin

Kraft¹,

Diefenbach²,

Mehta

et al. 1999

Journal of Biological Chemistry

130

128

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Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including ␣v␤6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin ␣v␤6. We compared the recognition profiles of recombinant ␣v␤6 and ␣v␤3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to ␣v␤3 contained ubiquitous RGD sequences. However, on ␣v␤6, in addition to RGD-containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to ␣v␤6 in isolated receptor assays (IC 50 ‫؍‬ 20 nM), and in cell adhesion assays (IC 50 ‫؍‬ 50 M). DLXXL peptides were highly specific inhibitors of ␣v␤6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH 2 -and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with ␣v␤6. The DLXXL sequence is present in several matrix components and in the ␤ chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin ␣v␤6 which has been unrecognized previously.Integrins are a family of heterodimeric class I transmembrane receptors involved in numerous cell-matrix and cell-cell adhesion phenomena (1). They can be grouped roughly into three classes: the ␤1 series, which are ubiquitous receptors for extracellular matrix (2); the ␤2 series, which are activatable on leukocytes and are triggered during the inflammatory response (3); and the ␣v series, which bind and mediate the cell response to provisional extracellular matrices found during wound repair and other pathological processes (4).The integrins ␣5␤1 (5), ␣IIb␤3 (6), ␣8␤1 (7), ␣v␤1 (8), ␣v␤3(9), and ␣v␤6 (10) all bind the Arg-Gly-Asp-(RGD) peptide sequence in fibronectin, where it is presented in a constrained loop (11). Soluble RGD-containing peptides can inhibit the interaction of each of these integrins with fibronectin. However, to analyze the function of individual fibronectin receptors in a particular cellular environment it is useful to have more specific inhibitors, and a variety of inhibitory antibodies, modified peptides, and non-peptidic substances has been developed. However, as yet no inhibitor has been discovered specific for ␣v␤6. ␣v␤6 is a rare integrin, induced during repair processes in epithelia (10, 12). Its only known specificities are for fibronectin (10), where it can be the dominant receptor me...

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“…Ligands-The ligands fibronectin (24), vitronectin (25), and fibrinogen (26) were purified from human blood and biotinylated as described previously (27,28).…”

Section: Methodsmentioning

confidence: 99%

Definition of an Unexpected Ligand Recognition Motif for αvβ6 Integrin

Kraft¹,

Diefenbach²,

Mehta

et al. 1999

Journal of Biological Chemistry

130

128

View full text Add to dashboard Cite

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“…Sodium salts representing different rankings in the Hofmeister series were evaluated over a range of concentrations and pH values. Effects of glycine, which is reported to be an antibody solubilizing/ stabilizing agent but a precipitant of fibrinogen, were also evaluated [13][14][15][16]. Possible applications of the technique to formulation development, as well as its limitations, are discussed.…”

Section: Introductionmentioning

confidence: 99%

An adaptation of hydrophobic interaction chromatography for estimation of protein solubility optima

Gagnon¹,

Mayes²,

Danielsson

1997

Journal of Pharmaceutical and Biomedical Analysis

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This study describes and adaptation of hydrophobic interaction chromatography (HIC) that can be used to estimate protein solubility optima. The method does not support determination of absolute, e.g. quantitative solubility, however it does provide a basis for identifying the salt concentration, pH, or additive concentrations that support the highest relative solubility. The magnitude of a given salt's effects are consistent with its ranking in the Hofmeister series. IgG in solutions of strong 'precipitating' salts exhibits a classical salting-in/salting-out curve, described by a solubility minimum at low ionic strength, increasing to a well-defined maximum, and then losing solubility with further elevation of salt concentration. The direct effect of pH on protein solubility, as well as its indirect effect via modification of the ionic equilibria of dissolved salts, can also be tracked. Cooperative effects of solubility modifiers such as amino acids can likewise be assessed. The technique can be a useful tool in the development of liquid formulations for protein pharmaceuticals. © 1997 Elsevier Science B.V.

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“…Human fibrinogen (90% clottable) was purchased from KabiVitrum and chromatographed on lysine-Sepharose to remove plasminogen (18). Fibrinogen which was to undergo iodination was purified from plasma by the Kazal technique and was 98% clottable (19).…”

Section: Methodsmentioning

confidence: 99%