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Urokinase-type plasminogen activator receptor

Inactive Publication Date: 2003-02-06
DANO KELD +20
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This analysis, however, does not show the purity of the preparation as it does not detect unlabelled proteins that may be present in an amount that may be higher than that of the u-PAR.
In addition, it cannot be evaluated whether contaminating non-labelled proteins are present, and as only a part of the lane in the SDS-PAGE is shown, even an evaluation of whether contaminating labelled proteins are present is impossible.
It was not possible to decide whether this low activity was intrinsic or due to contamination (Petersen et al., 1988).
These studies did not allow a rigorous discrimination between an activation process occurring in solution or between surface-bound reactants.
Possibly the presence of the bulky PAI-1 molecule may pose a problem of steric hindrance.

Method used

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  • Urokinase-type plasminogen activator receptor
  • Urokinase-type plasminogen activator receptor
  • Urokinase-type plasminogen activator receptor

Examples

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example 1

[0264] Purification and Characterization of u-PAR

[0265] Materials and Methods

[0266] SDS-PAGE.

[0267] When not stated otherwise, SDS-PAGE was performed according to Laemmli, U. K., "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature 227: 680-682, 1970, using 6-16% gradient slab gels. Pretreatment of samples under nonreducing conditions was performed without boiling. When reducing conditions were used, the samples were boiled for 5 minutes in the presence of 20 mM DTT.

[0268] Phast-gel SDS-PACE was performed on a Phast gel apparatus (Pharmacia), using ready-made 10-15% gradient gels. Electrophoresis was performed according to the recommendations of the manufacturer. Silver staining was performed according to Heukeshoven and Dernick, 1988.

[0269] Tricine-SDS-PAGE of samples to be electroblotted for amino acid analysis or NH.sub.2-terminal amino-acid sequencing was performed in a Mini Protean II apparatus (BioRad) according to Schagger and von Jago...

example 2

[0333] Isolation and Identification of the Ligand Binding Domain of u-PAR

[0334] Methods

[0335] Enzymatic Degradation:

[0336] Affinity purified u-PAR was dialyzed against 0.1% acetic acid and lyophilized as described in Example 1. The freeze-dried material was redissolved it incubation buffer (0.05 M Tris / HCl, 0.05% CRAPS, pH 8.1) to yield a protein concentration of approx 25 .mu.g / ml. 9 .mu.l samples of this u-PAR solution were treated with chymotrypsin (Worthington; final concentrations ranging from 8-200 ng / ml), by addition of 1 .mu.l of the appropriate stock solution of the enzyme, dissolved in incubation buffer. The samples were incubated for 16 h at 37.degree. C. after which the degradation was stopped by addition of 0.5 .mu.l of 20 mM phenylmethylsulfonylfluoride, dissolved in dimethylsulfoxide. The samples were stored at -80.degree. C. until analysis.

[0337] Analysis:

[0338] Direct electrophoretic analysis was performed by Tricine SDS-PAGE (see example 1) on a 10% T, 3% C gel aft...

example 3

[0350] Cloning of u-PAR

[0351] cDNA Libraries Used

[0352] A human cDNA library was used made from SV 40 transformed human GM637 fibroblasts in a plasmid vector based on pBR322 (carrying an ampicillin resistance gene) (Okayama H, Berg P, "High-efficiency cloning of full-length cDNA", Mol. Cell. Biol. 2: 161-170, 1982). The library was kindly donated by Dr. Okayama. This library was selected on the basis of the known high number of u-PAR in GM637 cells (Blasi, unpublished).

[0353] The plasmid vector (FIG. 8) uses the SV 40 promoter and has high expression in various eukaryotic cells, but very low or no expression in prokaryotes.

[0354] Screening Procedures

[0355] The library was screened with synthetic oligonucleotide probes made on the basis of amino acid sequence data from purified receptor protein (Tables 4-5). The melting temperatures were calculated from Lathe, J. Mol. Biol. 183: 1-12, 1985. The equation used was modified from:

t.sub.m-16.6 logM+0.41(% G+C)+81.5

[0356] in which M is the...

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Abstract

A polypeptide presenting an epitope cross-reactive with an epitope of urokinase-type plasminogen activator receptor, and / or having uPA binding activity, is described.

Description

[0001] The present invention relates to a method for preventing or counter-acting localized proteolytic activity in a mammal, in particular a human, the method comprising inhibiting the activation of plasminogen to plasmin by preventing the binding of a receptor binding form of urokinase-type plasminogen activator (in the following termed u-PA) to a u-PA receptor in the mammal and thereby preventing the u-PA from converting plasminogen into plasmin; the invention also relates to a pure u-PA receptor (in the following termed u-PAR), to DNA coding for the u-PAR, to the production of u-PAR or parts thereof for use as a therapeutic or diagnostic component, to u-PAR antibodies and the production of u-PA receptor binding u-PA molecules for use as a therapeutic or diagnostic component. In a further aspect, the invention relates to the regulation of the activity of a receptor binding form of u-PA, the activation of pro-u-PA to u-PA by plasmin and the regulation of the number of u-PARs on th...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K38/18A61K38/49A61K38/55A61K39/395C07K14/705C07K16/28C12N9/72C12N15/12C12Q1/68G01N33/574G01N33/86
CPCA61K38/00A61K38/49A61K39/3955C07K14/705C07K16/28C07K16/2896C07K2316/96C07K2317/77C12N9/6462C12Q1/6886G01N33/57446G01N33/86G01N2333/9723C12Y304/21073C12Q2600/158C07K2317/76
Inventor DANO, KELDBLASI, FRANCESCOROLDAN, ANN LOURINGCUBELLIS, MARIA VITTORIAMASUCCI, MARIA TERESAAPPELLA, ETTORESCHLEUNING, WOLF-DIETERBEHRENDT, NIELSRONNE, EBBEKRISTENSEN, PETERPOLLANEN, JARISALONEN, EEVA-MARJATTASTEPHENS, ROSS W.TAPIOVAARA, HANNELEVAHERI, ANTTIMOLLER, LISBETH BIRKELLIS, VINCENTLUND, LEIF ROGEPLOUG, MICHAELPYKE, CHARLESPATTHY, LASZLO
Owner DANO KELD
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