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

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.
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PMID:Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus. 229 45

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

A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.
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PMID:Production of bone-resorbing activity and colony-stimulating activity in vivo and in vitro by a human squamous cell carcinoma associated with hypercalcemia and leukocytosis. 348 54

Peptides such as somatostatin (SS14), epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulin-like growth factors (IGF-I and IGF-II) are present in breast milk from various species, and their significance in the developing gastrointestinal tract has been suggested. Our recent studies have indicated that rat milk soluble fraction (RMSF) protects SS14 in the gastrointestinal lumen by inhibiting in vitro the luminal peptidolysis. In the present studies, we have shown that RMSF inhibited in vitro degradation by midjejunal luminal flushings of suckling rats of 125I-labeled somatostatin 14[Tyr11], EGF, TGF alpha, IGF-I and IGF-II, as well as trypsin activity in vitro against benzoyl-L-arginyl-p-nitroanilide. The inhibitory factors present in the RMSF were further fractionated by gel filtration on Sephadex G100, ion-exchange chromatography on DEAE-Sephadex, and fast protein liquid chromatography (FPLC). Gel filtration of Sephadex G100 separated RMSF into three peaks of proteins: G1, G2, and G3; peptidase inhibitor activities were present exclusively in G1. Ion-exchange chromatography on DEAE-Sephadex column resolved peptidase inhibitory activity (G1) into three different peaks, D1, D2, and D3, eluted at sodium chloride concentrations of 0.05 M, 0.1 M, and 0.2 M, respectively. Further purification of D2 by FPLC resulted in a fraction rich in peptidase inhibitory activity, which was essentially free of trypsin inhibitory activity. Results indicate the presence of at least three peptidase inhibitors in rat milk, which may play a role in the protection of milk-borne peptides in the gastrointestinal lumen.
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PMID:Presence of multiple forms of peptidase inhibitors in rat milk. 814 98

The mechanism of regulation of mucus production in the gastric mucosa remains unclear. Recently, we established a gastric surface mucous cell line GSM06, which produces periodic acid-Shiff (PAS)-positive glycoconjugate (mucus) layers on the cell surface, from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. In this study, GSM06 cells were examined for its production of PAS-positive glycoconjugate layers to acid secretagogues and growth factors. The cells were cultured at nonpermissive temperature (39 degrees C) for 3-18 days and stained with PAS. Insulin (1-30 microg/ml; 0.29-8.6 microM) time- and dose-dependently increased production of glycoconjugates on the cell surface. When glycoconjugate layers produced by stimulation of insulin (3-30 microg/ml; 0.86-8.6 microM) were removed from the cell surface of GSM06 cells by a mild trypsin treatment, PAS-positive materials were remarkably decreased (day 18). In addition, morphological findings indicate that a high concentration of insulin (30 microg/ml; 8.6 microM) produced thick PAS-positive glycoconjugate layers just like normal gastric surface mucosa on the cell surface on day 18. In contrast, histamine (0.1-100 microM), carbachol (0.1-100 microM), gastrin-17 (0.1-100 nM), epidermal growth factor (0.01-10 ng/ ml; 1.7-1,700 pM), transforming growth factor-alpha (0.01-10 ng/ml; 1.8-1,800 pM), and fetal bovine serum (1-10%) did not increase glycoconjugate production. These findings suggest that insulin is a stimulator of glycoconjugate production, and stimulates production of glycoconjugate layers on the cell surface in the gastric surface mucous cell line GSM06.
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PMID:Insulin stimulates production of glycoconjugate layers on the cell surface of gastric surface mucous cell line GSM06. 901 7

Human breast cancer cells synthesize and release a variety of growth-modulating substances in response to estrogen stimulation, and it is generally accepted that the growth-promoting effects of estrogens are due at least in part to this autocrine/paracrine mechanism. Several of these growth-modulating substances, including transforming growth factor-alpha (TGF alpha) and its analogs, have been shown to require pericellular proteolysis for activation or release. Recently, we reported that MCF-7 human breast cancer cells are able to synthesize alpha 1-antitrypsin (alpha 1-AT), the major elastase inhibitor in human serum, and that there is a negative correlation between anchorage-independent growth of MCF-7 cells in soft agar and synthesis of alpha 1-AT. The studies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGF alpha from its membrane-bound precursor on MCF-7 cells is blocked by alpha 1-AT whether the cells were maintained in the presence or absence of estradiol and that there is a clear dose-response relationship between the alpha 1-AT concentration and both the release of TGF alpha and growth in soft agar. Consistent with this, TGF alpha release was increased in the presence of antibody to alpha 1-AT. In contrast, TGF alpha release and growth in soft agar were not blocked by peptide inhibitors specific for trypsin- or chymotrypsin-like enzymes. The alpha 1-AT concentration required for a half-maximal effect is lower for inhibition of TGF alpha release than it is for inhibition of colony formation (0.4 vs. 1.5 mumol/L). However, both values are in the range of concentrations one might expect at the cell surface in vivo. A new MCF-7 cell subline producing 10-fold higher levels of alpha 1-AT than its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of alpha 1-AT) with an alpha 1-AT complementary DNA. Growth in soft agar and release of TGF alpha were significantly decreased in cells transfected with the alpha 1-AT complementary DNA compared to those in cells transfected with vector alone, although, TGF alpha expression was the same. The above observations support a model for growth regulation in human breast ductal epithelial cells in which growth factor activation and release are dependent on the coordinate action of proteases and protease inhibitors. This model would predict that alpha 1-AT can act as a tumor suppressor in inhibiting the growth of breast cancer cells.
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PMID:Alpha 1-antitrypsin blocks the release of transforming growth factor-alpha from MCF-7 human breast cancer cells. 906 76

This study was performed to define the biologically active growth modulators in human gastric juice. Mitogenic activity was evaluated by the incorporation of [3H]thymidine into 3T3 fibroblasts. A negative correlation was observed between pH and mitogenic activity in gastric juice (r = -0.45, P < 0.01). The concentrations of epidermal growth factor (EGF), transforming growth factor-alpha and -beta 1 (TGF-alpha and -beta 1), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) in gastric juice did not explain these changes in mitogenic activity. Gel filtration identified growth-stimulating activity due to small molecule mitogens (less than 13 kDa), and growth inhibitory activity only in neutral samples due to a macromolecular substance (larger than 240 kDa) susceptible to trypsin digestion and heat and acid treatments. We conclude that acidity-dependent changes in mitogenic activity observed in this study are due to appearance of acid-unstable, high-molecular-weight, growth-inhibitory substance.
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PMID:Mitogenic properties of human gastric juice. 928 44

We reported previously that two epidermal growth factor receptor ligands, epidermal growth factor and transforming growth factor-alpha, inhibit medial septal cholinergic cell phenotypic expression (choline acetyltransferase and acetylcholinesterase activities) in vitro indirectly via (a) soluble molecule(s) released from astrocytes [Kenigsberg R. L. et al. (1992) Neuroscience 50, 85-97; Kenigsberg R. L. and Mazzoni I. E. (1995) J. Neurosci. Res. 41, 734-744; Mazzoni I. E. and Kenigsberg R. L. (1996) Brain Res. 707, 88-99]. In the present study, we found that this response to transforming growth factor-alpha is mediated, for the most part, by alpha 2-macroglobulin, a potent protease inhibitor with a wide spectrum of biological activities. In this regard, the effects of transforming growth factor-alpha on cholinergic cells can be blocked with immunoneutralizing antibodies raised against alpha 2-macroglobulin. Furthermore, western blot analysis reveals that although alpha 2-macroglobulin is present in conditioned media from control septal cultures, it is more abundant in those treated with transforming growth factor-alpha. In addition, exogenous alpha 2-macroglobulin, both in its native and trypsin-activated forms, can mimic transforming growth factor-alpha's effects on septal cholinergic cell expression. However, while the native antiprotease can slightly but significantly decrease choline acetyltransferase activity, trypsin-activated alpha 2-macroglobulin, in the nanomolar range, induces as marked a decrease in this enzyme activity as that noted with transforming growth factor-alpha. Furthermore, trypsin-activated alpha 2-macroglobulin, like epidermal growth factor/transforming growth factor-alpha, decreases choline acetyltransferase activity by arresting its spontaneous increase that occurs with time in culture, does so in a reversible manner and is not neurotoxic. In addition, trypsin-activated alpha 2-macroglobulin, in the nanomolar range, can affect choline acetyltransferase in a dual manner, up-regulating it at low concentrations while down-regulating it at higher ones. These responses are identical in mixed neuronal-glial and pure neuronal septal cultures. Furthermore, when concentrations of trypsin-activated alpha 2-macroglobulin, which alone decrease choline acetyltransferase, are added simultaneously with nerve growth factor, they serve to potentiate the nerve growth factor-induced increase in enzymatic activity. As GABAergic cell expression is not affected by alpha 2-macroglobulin, it appears that the effects of this protease inhibitor on medial septal neuronal expression are neurotransmitter-specific. Finally, trypsin-activated but not native alpha 2-macroglobulin promotes a dose-dependent aggregation of the septal neurons. This change in morphology, however, is not related to those noted in choline acetyltransferase activity. In summary, these data suggest that the expression of alpha 2-macroglobulin in astroglia from the medial septal nucleus can be controlled by epidermal growth factor receptor ligands to impact the functioning of basal forebrain cholinergic neurons.
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PMID:Transforming growth factor-alpha's effects on astroglial-cholinergic cell interactions in the medial septal area in vitro are mediated by alpha 2-macroglobulin. 933 Mar 64

Tripe palms are thickened, moss-like or velvety textured exaggerations of the normal dermatoglyphics. The disease belongs to the spectrum of papulosquamous paraneoplastic syndromes. Although suspected, the role of transforming growth factor-alpha (TGF-alpha) has not been clearly established. A 54-year-old man with systemic mastocytosis presented with thickening and darkening of the palms and soles. We performed skin biopsies for light microscopy (including toluidine blue), in situ hybridization and double labelling, and determination of serum tryptase, histamine and TGF-alpha levels. Toluidine blue stained the mast cells that had massively infiltrated the dermis. Tripe palm samples showed extensive hyperkeratosis. The TGF-alpha probe reacted strongly with the mast cells that also reacted with the antitryptase monoclonal antibody. Elevated tryptase, histamine and TGF-alpha levels prior to interferon-alfa administration decreased under treatment. The demonstration of TGF-alpha in infiltrating mast cells, the clinical regression of tripe palms and the lowering of the serum level and the mast cell molecular signal of the cytokine when systemic mastocytosis was controlled by interferon-alfa, suggest a key role for TGF-alpha in this cutaneous paraneoplastic syndrome.
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PMID:Tripe palms associated with systemic mastocytosis: the role of transforming growth factor-alpha and efficacy of interferon-alfa. 964 Mar 84

Growth of the myocardium involves the completion of a fixed number of rounds of cell division during the embryonic and fetal stages followed by entry into a postmitotic state and hypertrophy of the postmitotic cardiomyocytes during the later stages of heart growth. It has been suggested that at the time of its determination in the early embryo, the embryonic myocardium is programmed for a fixed and limited number of cell divisions, after which the transition to the postmitotic state occurs autonomously. The proliferative response of cultured myocardium of the fetal chick was explored in four culture settings: monolayer cell culture, collagen lattice culture, organ culture of reaggregated cardiomyocyte tissue (cardiomyocyte spheroid culture), and organ culture of pieces of the ventricle wall. Several growth factors were identified by their ability to stimulate DNA synthesis in cardiomyocytes, identified by the incorporation of 5-bromodeoxyuridine (BrdU). The serine proteases thrombin and trypsin, fibroblast growth factor-2 (FGF-2), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor-II (IGF-II) all show growth factor activity but only for cardiomyocytes cultured in three-dimensional myocardial tissue, and not for cardiomyocytes maintained in monolayer cell culture. Thus, during its proliferation phase, the growth of the fetal myocardium is not controlled solely by internal, cell-autonomous programs, but is subject to external regulation by a family of peptide growth factors. The unconventional setting of three-dimensional culture of the myocardium is required to demonstrate its responsiveness to growth factor challenge.
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PMID:Regulation of proliferation of the fetal myocardium. 1100 42


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