Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study demonstrates the existence on human peripheral blood lymphocytes of a saturable cell surface receptor for low density lipoprotein inhibitor (LDL-In), a subset of normal human serum low density lipoprotein (LDL) that has been previously demonstrated to suppress selected lymphocyte functions in vivo and in vitro. The binding of radioiodinated LDL-In of demonstrable biological activity occurs rapidly and is quantitatively augmented by prior cultivation of the lymphocytes in lipoprotein-depleted serum, suggesting regulation of receptor density by lipoproteins in vivo. Binding is temperature dependent, facilitated by calcium ions, saturable at 4 degrees C within 40-60 min, and blocked by prior exposure to unlabeled LDL-In. The lymphocyte receptor is trypsin sensitive and regenerates in vitro with a t1/2 of 3.6 h. LDL-In receptors are calculated to have a maximum density of 4,860 +/- 460 per cell if uniformly distributed on all lymphocyte subsets. These receptors have an estimated average association constant of 1.47 X 10(7) liters/mol. When considered in context of the estimated concentration of LDL-In in blood, the receptors should be partially occupied in vivo by endogenous plasma LDL-In. Prior site occupancy inhibition experiments designed to analyze the specificity of LDL-In binding demonstrate that (a) LDL-In is 13.7-fold more effective than whole LDL in blocking the subsequent binding of 125I-LDL-In to cells; and that (b) LDL is 11-fold more effective than LDL-In in blocking the binding of 125I-LKL. This is consistent with the degree of contamination of each lipoprotein with the other lipoprotein. An independent identity of the LDL-In receptor is also supported by observations that in contrast to the previously described LDL receptor, synthesis and expression of the LDL-In receptor on lymphocytes are not suppressed by cultivation of the cells in the presence of 25-hydroxycholesterol and cholesterol. These findings suggest the existence of a previously undescribed and discrete receptor on lymphocytes for LDL-In, and that the modulation of lymphocyte function by LDL-In may be mediated by a specific cell surface receptor pathway.
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PMID:Identification of a lymphocyte surface receptor for low density lipoprotein inhibitor, an immunoregulatory species of normal human serum low density lipoprotein. 20 35

Alveolar macrophages have been shown to bind glycoproteins and synthetic glycoconjugates (neoglycorpoteins) that have mannose, N-acetylglucosamine, or glucose in the exposed, nonreducing position. Galactose-terminal glycoproteins are not bound. Binding of radiolabeled ligands to cells is nearly completely impaired by the presence of an excess of yeast mannan. Binding is temperature sensitive and proceeds optimally at pH 7.0. Prior treatment of the cells with trypsin severely decreases their capacity to bind ligands. An inhibition assay has been developed, using radioiodinated glucose-albumin conjugate, agalacto-orosomucoid, beta-glucuronidase, and RNase B as ligands. Various glycoproteins have been shown to be effective inhibitors of ligand binding including horseradish peroxidase, agalacto-orosomucoid, beta-glucuronidase, ovalbumin, agalacto-fetuin, and RNase B. RNase A and asialo-fetuin are ineffective as antagonists. The results suggest the presence of a cell surface receptor on alveolar macrophages that binds glycoproteins having terminal sugars with the mannose or glucose configuration.
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PMID:Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. 27 29

We have used purified, (125)I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism-designated cbl C-in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4 degrees C reached a plateau after 3-4 hr; 95% of the bound (125)I was releasable with trypsin. Binding of TC II-Cbl at 4 degrees C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at congruent with5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'- tetraacetic acid. At 37 degrees C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound (125)I. After 2 h at 37 degrees C, increasing amounts of acid-soluble (125)I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.
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PMID:Binding and uptake of transcobalamin II by human fibroblasts. 61 8

Studies were designed to evaluate the binding of binding of vitamin B12 to cell membrane preparations from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin B12 alone, binds to a single saturable binding site with an approximate Ka = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pretreating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the binding site includes a relatively exposed membrane protein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton X-100.
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PMID:A saturable high affinity binding site for transcobalamin II-vitamin B12 complexes in human placental membrane preparations. 83 Jun 65

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.
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PMID:Functional characterization of protease-treated Bacillus anthracis protective antigen. 151 56

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.
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PMID:Tumor-associated urokinase-type plasminogen activator: biological and clinical significance. 151 91

Cyclosporin A (CsA) is an immunosuppressive agent that inhibits the synthesis of lymphokines by T lymphocytes at the level of transcription. A cytoplasmic protein, cyclophilin, is the most thoroughly studied CsA-binding protein, but its ubiquitous presence in cells of all types raises questions about its role in immunosuppression. In an attempt to ascertain the presence of a cell surface receptor, we synthesized two polyvalent macromolecular CsA derivatives, CsA-BBa-ovalbumin and CsA-BBa-aminodextran (CBD), from the product of the photochemical reaction of CsA and 4-benzoylbenzoic acid (CsA-BBa). (i) They inhibited the peptidylprolyl cis-trans isomerase activity of cyclophilin and the synthesis of interleukin 2 by phorbol ester-activated EL-4 cells. (ii) CBD also inhibited interleukin 2 secretion by Con A-activated T-cell-enriched mouse splenocytes. 4-Benzoylbenzoic acid (BBa)-aminodextran and aminodextran were inactive. (iii) Direct binding and competition studies with [3H]CsA indicated that CBD does not enter EL-4 cells (i.e., it acted at the surface). (iv) CBD caused agglutination of EL-4 cells, murine B and T lymphocytes, human thymocytes, and two T-cell hybridomas. Agglutination was inhibited by a monoclonal antibody to CsA and by CsA and CsA-BBa, but not by BBa. No agglutination was seen with BBa-aminodextran or aminodextran. HeLa cells, Vero (monkey kidney) cells, a mouse plasmacytoma, COS cells, and a poorly differentiated B-cell lymphoma were not agglutinated. (v) EL-4 cells failed to be agglutinated after treatment with trypsin or chymotrypsin. Specific agglutination was again possible after incubation for 5 h at 37 degrees C in the absence of enzyme. (vi) CBD covalently linked to crosslinked agarose beads inhibited interleukin 2 production by phorbol ester-stimulated EL-4 cells. No activity was seen if cell-to-bead contact was prevented by a 0.02-microns microporous filter that did not interfere with the passage of CBD. Our findings support the presence of a functional receptor on the surface of selected cells of the immune system.
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PMID:Evidence for a functional receptor for cyclosporin A on the surface of lymphocytes. 158 69

Formation of the covalently stabilized alpha 1-antitrypsin (alpha 1-AT)-neutrophil elastase complex, the archetype of serpin-enzyme complexes, results in a structurally rearranged alpha 1-AT molecule that possesses chemo-attractant activities, mediates an increase in synthesis of alpha 1-AT by mononuclear phagocytes and hepatocytes, and is more rapidly cleared from the circulation than is the native alpha 1-AT molecule. We have recently identified an abundant, high affinity cell surface receptor on human hepatoma HepG2 cells and human monocytes that binds alpha 1-AT-elastase complexes, mediates endocytosis and lysosomal degradation of alpha 1-AT-elastase complexes, and induces an increase in synthesis of alpha 1-AT. We have referred to this receptor as the serpin-enzyme complex, or SEC, receptor because it also recognizes complexes of serpins antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor with their cognate enzymes. In the current study, we show that a pentapeptide domain in the carboxyl terminal fragment of alpha 1-AT (amino acids 370-374, FVFLM) is sufficient for binding to the SEC receptor. A synthetic analog of this pentapeptide (peptide 105C, FVYLI) blocks binding and internalization of alpha 1-AT-125I-trypsin complexes by HepG2 cells. 125I-Peptide 105C binds specifically and saturably to HepG2 cells, and its binding is blocked by alpha 1-AT-trypsin or alpha 1-AT-elastase complexes. Alterations of this sequence introduced into synthetic peptides (mutations, deletions, or scrambling) demonstrate that binding of the pentapeptide domain is sequence-specific. Comparisons with the sequences of other serpins in the corresponding region indicate that this pentapeptide neodomain is highly conserved.
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PMID:The SEC receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease complexes. 164 29

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.
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PMID:Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins. 164 6

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.
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PMID:A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity. 183 Nov 99


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