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)

A kininogenin (EC 3.4.21.8) was purified from the venom of Vipera ammodytes ammodytes (European sand viper) by a combination of gel filtration and ion-exchange chromatography. The enzyme is approximately six times more active than bovine trypsin in its ability to release vasoactive peptides from a plasma precursor. The kininogenin is a glycoprotein containing 18-20% by weight of carbohydrate. It showed a mol. wt. of 40500 on gel filtration. Gel electrophoresis of the reduced sample in the presence of sodium dodecyl sulphate and 2-mercaptoethanol revealed the presence of two major components of mol.wt. 34300 and 31300. The heterogeneity, which was also observed on disc electrophoresis, was removed by incubation with neuraminidase. After incubation with neuraminidase the kininogenin retained full enzymic activity and possessed an isoelectric point of pH7.2. The carbohydrate content has been decreased to 10% by weight, and the single component seen on electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol corresponded to a mol.wt. of 29500.
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PMID:Purification and properties of a kininogenin from the venom of Vipera ammodytes ammodytes. 127 96

We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation. Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138-139 days, either ACTH(1-24) (10.5 micrograms/0.24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0.9% (w/v) NaCl alone (0.24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0.2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2.0 x 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H2O2, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of fetal hypophysectomy with or without ACTH replacement on the molecular weight profile of enkephalin-containing peptides in the adrenal medulla of the fetal sheep. 132 54

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.
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PMID:Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates. 135 Jul 84

Interleukin-1 (IL-1) is an important mediator in inflammation and immunological processes. The findings of native IL-1 inhibitors suggest a negative feedback mechanism to down-regulate IL-1 mediated acute inflammation. IL-1 inhibitors were also found elevated in disease states associated with high IL-1 levels. We have previously described one such IL-1 inhibitor derived from the human M20 myelomonocytic cell line. In this paper we present several biological and biochemical characteristics of the M20 IL-1 inhibitor. Various in vitro activities of the inhibitor are described and its IL-1 specificity in these assays is demonstrated. Purification of the inhibitor was performed by DEAE-high performance liquid chromatography, isoelectric focusing, gel filtration and dye ligand chromatography column. This protein factor has a MW of 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. The inhibitor has no cross-reactivity against a panel of known cytokines (IL-1 alpha, IL-1 beta, IL-2, sIL-2R, IL-6, tumor necrosis factor (TNF), interferon-gamma (IFN-gamma)) and is distinct from the IL-1 receptor antagonist (IL-1ra). The purified IL-1 inhibitor was destroyed by trypsin, 2-mercaptoethanol, sodium dodecyl sulfate and extremes in pH and in temperature. Only IL-1 induced (but not the IL-2, IL-6 or TNF induced) thymocyte proliferation and PGE2 production by fibroblasts were inhibited by the inhibitor, thus showing specificity to IL-1 in these assays.
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PMID:The M20 IL-1 inhibitor. II. Biological characterization. 143 Nov 47

The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures. Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay. Recombinant HBcAg retained its immunoreactivity when heated at 70 degrees C for 60 min but was inactivated at 85 degrees C in 10 min. It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5. Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity. The proteolytic enzymes papain and bacterial protease (type VIII from Bacillus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not. The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50% of the initial level. This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.
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PMID:Stability of the recombinant hepatitis B core antigen. 162 88

Pseudorabies virus hemagglutinin was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on cattle erythrocytes. The adsorbed hemagglutinin could not be eluted from the cells by resuspending in phosphate-buffered saline (PBS), by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The receptor on mouse erythrocytes for the hemagglutinin was inactivated by trypsin, but not by neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin. The hemagglutinin was inactivated by trypsin, alpha-amylase, pepsin, DOC, KIO4, and ethylendiamine-tetraacetic acid (EDTA), but not by papain, beta-glucosidase, phospholipase C, neuraminidase, DTT, 2-ME, Tween-80, ethylether, chloroform, trichloro-trifluoroethane, beta-propiolactone and formalin, suggesting that the hemagglutinin active component involved glycoproteins. The hemagglutinin was stable at 37 degrees C for lower temperatures but not at 60 degrees C or higher. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 48,000 x g for 3 hr. In rate zonal centrifugation of the preparation on a sucrose density gradient, the hemagglutination (HA) activity showed a sharp peak at 1.22 g/ml coinciding with the peak of infectivity. The HA activity in the peak fraction seemed to be structually associated with virus particles. After fractionation of the virus by Nonidet P-40, the HA activity was found only in the fraction of the envelope material, indicating that the hemagglutinin is situated in the viral envelop.
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PMID:Physicochemical properties of pseudorabies virus hemagglutinin. 166 85

Using transmission electron microscopy, gold-labeled lectins, morphometry and enzyme-linked lectin assay, we could show that treatment of promastigotes of Leishmania donovani chagasi with trypsin did not interfere with the binding of lectins (concanavalin A, peanut agglutinin, wheat germ agglutinin and Ricinus communis agglutinin) to the parasite surface. These observations are in agreement with results we previously obtained using a biochemical approach. Treatment of fixed promastigotes with 2-mercaptoethanol induced a significant increase in the density of concanavalin A (Con A) receptors on the surface of L. d. chagasi in relation to the control. We suggest that this increase is due to the unfolding of one or more surface glycoproteins after cleavage of disulfide bonds between cystein residues in adjacent protein loops, exposing second-order Con A receptors that are otherwise hidden in the protein quaternary structure.
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PMID:Effect of trypsin and 2-mercaptoethanol on the exposure of sugar residues on the surface of Leishmania donovani chagasi. 179 23

Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.
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PMID:Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant. 200 12

An improved method for the purification of human placental transferrin receptor (Tf-R) was developed. Fresh human placenta was homogenized in cold acetone and the acetone powder was prepared. After the acetone powder had been washed with HEPES buffer, the insoluble proteins containing Tf-R were separated by centrifugation and dissolved in Emulgen 109P-containing buffer. Tf-R was collected by affinity binding to Tf-Sepharose and extracted by consecutive treatment with 4 different kinds of buffers. Tf-R was eluted by buffer C (2 M KCl) and buffer D (0.5 M NaSCN). Tf-R was characterized as a 90-kDa monomer on gradient SDS-PAGE (4-20%) in the presence of 2-mercaptoethanol. Though there were several minor bands of 180- and above 205-kDa, all these bands were confirmed as Tf-R by Western blotting using an anti-Tf-R monoclonal antibody (OKT 9). The apparent molecular weights, measured by HPLC using a TSK-G 3,000 SW column, demonstrated that Tf-Rs eluted with buffer C were approximately 370-, 500- and above 500-kDa, but only a peak of above 500-kDa was found in Tf-R eluted with buffer D. Although the polymers of Tf-R with molecular weight of above 500-kDa were resistant to trypsin digestion, the Tf-R of 370-kDa was resistant to the enzyme only when it conjugated to the diferric Tf. The stability of the polymers of above 500-kDa to trypsin digestion suggested an advantage for the repeated use of Tf-R in the endocytosis of diferric Tf, which was performed by the translocation of Tf-R between cell surface and intracellular vesicles.
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PMID:[An improved purification of human placental transferrin receptor and biochemical properties of the receptor]. 206 90

The release of Zn2+ from transcription factor IIIA (TFIIIA) was examined with the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) in the absence and presence of p-hydroxymercuriphenylsulfonate (PMPS). With 0.5 mM PAR, approximately 5 eq of Zn2+ were released from TFIIIA, but no Zn2+ release was detected from the 7 S ribonucleoprotein. The PMPS-promoted Zn2+ release from TFIIIA was 8.7 +/- 0.4 eq of Zn2+ of which approximately 4 eq of Zn2+ rebound to TFIIIA upon displacement of the mercurial with excess 2-mercaptoethanol. These results suggest that at least two affinity classes of Zn2+ binding sites exist in TFIIIA, one of which is released to 0.5 mM PAR in the absence of PMPS. Also, 18 of the 23 cysteine residues of TFIIIA reacted with 5,5'-dithiobis-(2-nitrobenzoic acid). The kinetic data of PAR and 5,5'-dithiobis-(2-nitrobenzoic acid) reactions with TFIIIA were similar, and the spectral changes were characterized by at least three exponential terms. Both TFIIIA and the 7 S particle were reacted with the thiol-specific fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS). Complete trypsin hydrolysis followed by reverse-phase high pressure liquid chromatography analysis of peptide mixtures showed only one fluorescent peak from the AEDANS-labeled 7 S particle whereas numerous fluorescent peaks were observed with AEDANS-labeled TFIIIA. This further indicates exposure of cysteine residues from Zn2+ binding domains in TFIIIA. Cys287 was identified as the site of modification by amino acid sequencing of the isolated fluorescent peptide from the derivatized 7 S particle. Limited papain cleavage of the AEDANS-labeled 7 S particle indicated that the modified cysteine is located within a 34-kDa TFIIIA fragment. Gel retardation and transcription assays showed that TFIIIA, which had been purified from the AEDANS-labeled 7 S particle, was capable of binding to the internal control region of 5 S RNA gene and retained transcription activity. Thus, Zn2+ binding domains and all but 1 cysteine residue are buried in the 7 S particle, thereby facilitating site-specific labeling of TFIIIA.
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PMID:Xenopus transcription factor IIIA. Evidence for heterogeneity of Zn2+ binding affinities and specific labeling of cysteine 287. 211 9


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