Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.
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PMID:Urokinase binding and receptor identification in cultured endothelial cells. 165 68

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC lead to an increase in its apparent molecular mass by approximately 40 Kd. The predominant u-PA binding protein isolated from whole cell detergent extracts migrated with a molecular mass of approximately 36 Kd using affinity chromatography. In contrast, when only cell surface proteins were radiolabeled before extraction, the predominant labeled u-PA binding protein isolated migrated with a molecular mass of approximately 46 Kd. Several pieces of evidence suggested that the difference in molecular mass between these two u-PA binding proteins resulted from glycosylation of a single receptor protein. First, a polyclonal antibody against u-PA receptor isolated from phorbol myristate acetate (PMA) stimulated U-937 cells reacted with both the 36- and 46-Kd proteins on Western blotting. Second, the size of the unmodified receptor was estimated by amplifying a full-length cDNA for u-PA receptor from an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence of a single 1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of 35S-labeled 46-Kd cell surface receptor protein was inhibited when the cells were grown in the presence of tunicamycin, while the synthesis of the 36-Kd species was unaffected. Moreover, the apparent molecular mass of purified surface-labeled receptor (approximately 46 Kd) was reduced by N-glycanase. These studies suggest that the u-PA receptor on the surface of HUVEC is a glycoprotein derived from a protein of approximately 35 Kd which is similar immunologically to u-PA receptors on other cell types.
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PMID:Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA. 217

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.
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PMID:The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1. 251 81

The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures. The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae.
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PMID:The major N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated, (alpha 2-6)-sialylated or (alpha 1-3)-fucosylated N-acetylgalactosamine(beta 1-4)-N-acetylglucosamine elements. 773 45

uK2t-PA is a hybrid plasminogen activator in which the epidermal growth factor-like domain of the urokinase-type plasminogen activator precedes the kringle 2 and catalytic domains of tissue-type plasminogen activator. The molecules are expressed in Chinese hamster ovary cells in two variant forms, a type II form in which only the protease domain is glycosylated, and a type I form in which both the kringle 2 and the protease domains carry N-acetyllactosamine type glycans. The two forms differed slightly in their affinity for fibrin and fibrinogen, which allowed their separation, but the stimulation of plasminogen activation of the type II form by fibrin was up to eight-fold lower than that of the type II form. The sensitivity to fibrin could be restored by treatment of the type I form with N-glycanase or sialidase. Enzymatic activity vs low molecular weight substrates was not influenced by the glycosylation of kringle 2.
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PMID:Functional effects of kringle 2 glycosylation in a hybrid plasminogen activator. 838 98

Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed by N-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4-6-fold increase in Kd) as assessed by real time biomolecular interaction analysis.
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PMID:Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells. 959 42