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)

Trypsin treatment of recombinant PDGF-BB from Escherichia coli leads to the liberation of a small carboxy-terminal fragment and two internal segments without dissociating the molecule. The remaining core of 21 kDa retained a considerable binding affinity of 8.4 nM. By use of various peptide fragments obtained from monomeric recombinant PDGF-B, a receptor binding domain was assigned to one of these internal trypsin-sensitive segments. This segment is enriched in charged residues, suggesting mainly hydrophilic interactions with the receptor. Circular dichroism measurements of recombinant PDGF-BB showed a high content of random structure and only a small percentage (less than 10%) of alpha-helical structures. This structure was very rigid since the addition of 70% trifluoroethanol or 1% SDS did not change the circular dichroism spectrum. On the basis of these results, a tentative structure was generated by computer modeling.
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PMID:Binding domains and epitopes in platelet-derived growth factor. 247 33

We have previously reported the presence of a high molecular weight polypeptide growth factor in the plasma of normal human or rat serum which stimulates DNA synthesis in primary cultures of normal rat hepatocytes. We referred to this activity as hepatopoietin A (HPTA) (Michalopoulos, G., Houck, K. A., Dolan, M. L., and Luetteke, N. C. Control of hepatocytes replication by two serum factors. Cancer Res., 44: 4414-4419, 1984; Thaler, J., and Michalopoulos, G. Hepatopoietin A. Partial characterization and trypsin activation of a hepatocyte growth factor. Cancer Res., 45: 2545-2549, 1985). At that time, however, complete purification of this growth factor had not been achieved. In the present report we describe the steps required for complete purification of HPTA from human plasma or rabbit serum. The purification involved sequential ammonium sulfate precipitation, heparin-affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and reversed phase HPLC. The final purified product is a heterodimer consisting of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Under nonreducing conditions, however, the purified HPTA migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 69,000. The mitogenic activity of HPTA was associated with this band when it was eluted from unstained sodium dodecyl sulfate-polyacrylamide gels. Gel filtration HPLC under neutral isotonic conditions indicated that HPTA tends to form aggregates with molecular weights of greater than 300,000. Chromatofocusing indicated that HPTA is an acidic protein with an isoelectric point value of about 5.5. The mitogenic activity of HPTA was sensitive to heat, trypsin, and 2-mercaptoethanol, but relatively resistant to exposure to 1 N acetic acid, 2 M guanidine-HCl, and 0.1% sodium dodecyl sulfate. The stimulation of DNA synthesis induced by HPTA was totally abrogated by transforming growth factor-beta and markedly reduced in the presence of heparin. We present biochemical as well as biological evidence that HPTA is a hepatocyte growth factor distinct from other known polypeptide mitogens such as epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, fibroblast growth factor, and thrombin.
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PMID:Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. 252 51

The effect of concentrated conditioned medium from each of eight human malignant glioma cell lines on the growth of indicator cells (normal rat kidney fibroblasts (NRK), clone 14) was determined in monolayer and in soft agar assay systems. The conditioned medium from all cell lines was mitogenic in the monolayer assay, but only SF-210, U-343 MG-A, and U-251 MG produced soluble factors that caused NRK cells to grow in soft agar. The soluble growth-promoting factors from these three cell lines were acid- and heat-stable (60 degrees C for 30 minutes) but were inactivated by trypsin (100 microns/ml) and dithiothreitol (50 microM). The growth factors from SF-210 and U-343 MG-A were further purified by molecular-sieve chromatography. The partially purified growth factor from U-343 MG-A retained transforming growth factor (TGF)-like activity, had a molecular weight of 9 kD, was potentiated by TGF-beta in the soft agar assay, competed effectively with 125I-epidermal growth factor (EGF) radiolabeled for the EGF receptor on A 431 epidermoid carcinoma cells, and was completely inhibited by monoclonal antibodies to TGF-alpha. The partially purified growth factor from SF-210 had a molecular weight of 17 kD, was not inhibited by monoclonal antibodies to platelet-derived growth factor (PDGF) or TGF-alpha, and did not bind to a heparin-Sepharose column. These results imply that U-343 MG-A secretes a growth factor with TGF-alpha-like activity, and SF-210 secretes a TGF with neither TGF-alpha nor TGF-beta activity.
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PMID:Isolation and partial purification of growth factors with TGF-like activity from human malignant gliomas. 258 80

Interactions of vascular endothelial cells (ECs) and smooth muscle cells (SMCs) were studied by testing the ability of cultured bovine aortic ECs to secrete factors influencing the migration of cultured aortic SMCs from the same species. Migration of SMCs was examined in blind-well chambers using gelatin-coated polycarbonate filters. Conditioned culture medium obtained by incubating confluent monolayers of ECs in serum-free RPMI-1640 medium for 48 hours caused a 2.4-fold increase in the migration of SMCs as compared with nonconditioned medium (p less than 0.001). The effect was dependent on the length of conditioning with the ECs and was chemotactic in nature as judged on the basis of checkerboard analysis. Preliminary characterization of the migration stimulating activity indicates that it is sensitive to trypsin, nondialyzable, and stable at 56 degrees C for 30 min. The activity was abolished by heating to 100 degrees C for 20 min but was not significantly inhibited by protamine sulphate, which suggests that most of the activity was not due to platelet-derived growth factor (PDGF)-like proteins. Our results thus show that ECs secrete polypeptide(s) chemotactic for vascular SMCs. Such interactions between ECs and SMCs in vivo might contribute to the migration of medial SMCs into the intima during atherogenesis.
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PMID:Cultured bovine aortic endothelial cells secrete factor(s) chemotactic for aortic smooth muscle cells. 271 9

Because platelet-derived growth factor (PDGF) may be released at sites where neutrophil proteinases may also be released, we examined the effects of neutrophil elastase and cathepsin G upon the chemotactic and mitogenic activities of PDGF. Elastase abolished the chemotactic activity of PDGF for fibroblasts but had no effect on its chemotactic activity for monocytes, or on its mitogenic activity for 3T3 cells or its capacity to bind to 3T3 cells. Cathepsin G had no effect upon the chemotactic or mitogenic activities of PDGF. In contrast, trypsin eliminated the chemotactic activity of PDGF for monocytes and fibroblasts and the mitogenic activity of PDGF. After reduction and alkylation, PDGF retained full chemotactic activity for fibroblasts and monocytes but exhibited no mitogenic activity and only limited binding to 3T3 cells. These results indicate separate domains on PDGF for fibroblast chemotactic and mitogenic activity and for monocyte and fibroblast chemotactic activity and raise the possibility that the biological activities of PDGF may be modified selectively in vivo. The findings further suggest that the majority of PDGF receptors on fibroblasts mediate mitogenic activity and that only a minority of the PDGF receptors on fibroblasts are responsible for chemotactic activity.
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PMID:Dissociation of the chemotactic and mitogenic activities of platelet-derived growth factor by human neutrophil elastase. 298 84

Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells.
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PMID:Proliferation of mouse uterine epithelial cells in vitro. 300 88

The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis. In the absence of exogenous PDGF, the apparent half-life of this pool was 2 h. The presence of both N- and O-linked oligosaccharide chains on cell surface PDGF receptors was demonstrated. Enzymatic removal of the N-linked oligosaccharide chains reduced the receptor's apparent Mr by approximately 40 kDa and removal of O-linked oligosaccharide caused approximately a 7-kDa reduction. Activation of receptor tyrosine autophosphorylation by PDGF did not require either processing of high-mannose N-linked oligosaccharides to complex forms or the presence of sialic acid on receptor oligosaccharide chains. Tryptic cleavage of PDGF-activated surface receptors in intact cells yielded two discrete phosphotyrosine-containing fragments of 107 and 85 kDa. Cleveland digest patterns from each fragment indicate that both are derived from the intact PDGF receptor. These data indicate that PDGF receptors are synthesized and turn over rapidly in the absence of ligand. Partial characterization of the extracellular domain oligosaccharide contribution to receptor function and trypsin susceptibility is provided.
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PMID:Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors. 303 71

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.
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PMID:Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells. 336 10

Cementum forms the interface between root dentin and periodontal ligament through which periodontal connective tissue is attached to root surfaces. We have examined how cementum components influence the biological activities of gingival fibroblasts. Cementum was harvested from freshly extracted human teeth and extracted sequentially with 0.5 mol/L acetic acid, 4 mol/L guanidine-0.5 mol/L EDTA, and bacterial collagenase. The extracts were concentrated and analyzed for mitogenic activity to human gingival fibroblasts. DNA synthesis was assayed by measurement of [3H]thymidine incorporation by quiescent fibroblasts activated to divide, and cell growth was determined by the counting of cells over a 10-day period. Results showed that extracts of cementum stimulated quiescent gingival fibroblasts to synthesize DNA and grow. The stimulation was dose-dependent, and most of the stimulatory activity was extracted by acid. Addition of small quantities of serum potentiated the mitogenic activity to levels greater than those of control cultures containing 10% fetal calf serum. The mitogenic activity was heat-stable, but it was destroyed by trypsin. Neither platelet-derived growth factor (PDGF) nor epidermal growth factor (EGF) was detectable in the cementum extract, and extracts of human dentin and skin contained very little mitogenic activity. We conclude that cementum contains substances capable of regulating the growth of gingival fibroblasts, and that these substances may play an important role in gingival connective tissue formation and regeneration.
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PMID:Mitogenic activity of cementum components to gingival fibroblasts. 347 10

Skeletal growth factor (SGF) activity was extracted from human bone matrix by demineralization and purified under dissociative conditions using hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was characterized chemically and biologically. Purified human SGF stimulated chick embryo bone cell proliferation at picomolar concentrations (half maximum at 2-3 ng/ml) and had little or no activity on other cell types tested (mouse 3T3 and normal rat kidney fibroblasts, embryonic chick intestinal and human placental cells). Human SGF did not displace 125I-labeled epidermal growth factor binding to normal rat kidney cells and did not stimulate normal rat kidney cell colony formation in soft agar. Human SGF activity was sensitive to trypsin, chymotrypsin, papain, dithiothreitol and performic acid but was resistant to heat (upto 70 degrees C), pH (3-10), cyanogen bromide, alkaline phosphatase and neuraminidase and did not bind jack bean concanavalin A or kidney bean lectin. From our chemical and biological studies it appears that human SGF is different from other known polypeptide growth factors: epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor.
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PMID:Chemical and biological characterization of low-molecular-weight human skeletal growth factor. 349 Feb 78


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