Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Two monoclonal antibodies, M32-1 (Ig-G1,k) and M39-2 (IgM,k), were prepared against high molecular weight (greater than 650 kDa) cytosol antigens (HMW-CA) of a human adenocarcinoma of the colon (GW-39). These monoclonal antibodies appeared to bind to determinants on two distinct high molecular weight colon antigens. One was shown by gel filtration to be a 650 kDa glycoprotein (gp650) containing at least one 300 kDa antigenic subunit (gp300). The other antigen eluted from a S-300 Sephacryl column at a molecular size of 600 kDa (gp600) and was resistant to dissociation by detergents, salts and chaotropic agents. The differential sensitivity of these two high molecular weight glycoproteins to treatment with trypsin, chondroitinase ABC, HNO2, endoglycosidase H and 2-mercaptoethanol suggest that monoclonal antibodies M39-1 and M39-2 react with distinct antigenic determinants located on two separate, high molecular weight, colon antigens. Since these antigens are only detected in extracts prepared from normal mucosa, well-differentiated tumors or margins of well-differentiated tumors, their expression appears to be related to a well-differentiated cell phenotype.
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PMID:Characterization of two monoclonal antibodies that recognize high molecular weight colon antigens. 292 Mar 72

Adult Brugia malayi nematode parasites possess a range of cuticular and epicuticular molecules which may be defined by various surface-labelling techniques. We present here evidence that at least two distinct antigens are associated with the surface, a glycoprotein of 29 kDa, and a complex of twelve components forming a regular series or 'ladder' between 17 and 200 kDa (17/200 kDa) which have not previously been described. Each of these products is antigenic in infected hosts, although responses in infected humans to the 17/200 kDa are relatively weak. Digestion of the 29 kDa antigen with proteases and endoglycosidases indicates that it is closely conserved between B. malayi and B. pahangi, and that it carries at least two N-linked oligosaccharide chains each of 1.5-2 kDa. By contrast, a smaller surface-labelled antigen of 15 kDa shows no glycosylation by either lectin adherence or endoglycosidase digestion assays. Trypsin treatment of intact, labelled parasites results in cleavage of 29 kDa molecules isolated 17/200 kDa 'ladder' to trypsin abolishes all bands except the 17 kDa base unit. Both the 29 kDa and 17/200 kDa antigens can be recovered as water-soluble molecules by homogenisation of the parasite in the absence of detergent, or by disruption of the cuticle with reducing agents such as 2-mercaptoethanol. In the presence of such agents, both the 17/200 kDa series and the 29 kDa glycoprotein are shed rapidly from intact parasites. Finally, two-dimensional electrophoretic analysis shows that while the 29 kDa glycoprotein is strongly basic and the 15 kDa acidic, the 17/200 kDa antigens form a related series of neutral pI.
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PMID:Filarial surface antigens: the major 29 kilodalton glycoprotein and a novel 17-200 kilodalton complex from adult Brugia malayi parasites. 292 46

Dynein 1 was extracted from sperm flagella of the sea urchin Tripneustes gratilla with 0.6 M NaCl and dialyzed against 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 5 mM imidazole/HCl buffer, pH 7.0, for 24-48 h. In some cases, fractions containing the alpha heavy chain and the beta/intermediate chain 1 complex (beta/IC1) were separated by density gradient centrifugation in the same solution. Treatment of the samples at a trypsin:protein ratio of 1:10 w/w for 32 min at room temperature yields a crude digest from which Fragment A is purified by density gradient centrifugation. The purified Fragment A consists of two principal peptides (Mr = 195,000 and 130,000) that cosediment with the peak of ATPase activity at 12.5 S, which is slightly faster than the 11 S of the original beta/IC1 complex. When digests of the separated alpha chain and of the beta/IC1 complex are followed as a function of time, the early cleavages of the two heavy chains (Mr = 428,000) resemble each other in that both lead to similarly sized peptides of Mr 316,000 and 296,000, but only in the beta/IC1 fraction does the digestion proceed to form Fragment A. The remainder of the beta chain, termed Fragment B, occurs as an Mr 110,000 peptide sedimenting at 5.7 S with no associated ATPase activity. Fragment A has a specific ATPase activity of 4.3 mumol Pi X min-1 X mg-1, with a Km of 29 microM in 0.1 M NaCl medium, and an apparent Ki for inhibition by vanadate of 1.2 microM in the absence of salt, and 22 microM in 0.6 M NaCl. Photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-triphosphate indicates that the ATP binding site on the beta chain of dynein 1 is located on the Mr 195,000 peptide of Fragment A. The possibility that Fragments A and B of the beta/IC1 complex may correspond to the head and tail regions of the tadpole-shaped particle seen by electron microscopy is discussed.
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PMID:Tryptic digestion of dynein 1 in low salt medium. Origin and properties of fragment A. 295 97

Polar membrane in Campylobacter jejuni has been visualized on membrane vesicles. It was composed of doughnut-shaped particles 5-6 nm in diameter, with stalks, arranged in a hexagonal array. This structure was stabilized on the membrane by a high ionic strength buffer in the presence of 2-mercaptoethanol. Histochemical staining indicated localized ATPase activity at the poles of the cells. An ATPase with distinctive properties has been isolated and purified from this organism; it gives a specific activity of approximately 0.3 units/mg of protein. Electron microscopy showed doughnut-shaped particles 5-6 nm in diameter. Nondissociating and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed, respectively, a single band with ATPase activity and a molecular weight of ca. 75,000 Da. The enzyme was cold labile and activity was abolished by trypsin. Dicyclohexylcarbodiimide inhibited the membrane-bound form of the enzyme, but did not inhibit the soluble form. Oligomycin had no inhibitory activity on either form of the enzyme. The enzyme specifically hydrolysed ATP, but other nucleotide substrates were not degraded. The enzyme was activated by Mg2+ and inhibited by Ca2+, whereas other ions had no effect on activity. Antibodies prepared to this enzyme bound to the polar regions of whole cells as shown by protein A - colloidal gold immunoelectron microscopy. The antibodies to this ATPase cross reacted (shown by Western blotting) with four proteins from a whole-cell extract of this organism, two proteins in Aquaspirillum serpens MW5, and three proteins from Escherichia coli K12. They did not cross-react with any proteins from Spirillum volutans, Methanococcus voltae, Vibrio cholerae, or rat liver mitochondria. Antibodies raised against the F1-ATPase of E. coli K12 cross reacted with six proteins in a whole-cell extract of this organism, and one protein species in each of the whole-cell extracts of V. cholera, A. serpens MW5, S. volutans, and rat liver mitochondria. These antibodies did not recognize any whole cell proteins from either C. jejuni or M. voltae. These results along with the ATPase activity localized by histochemical staining suggest that polar membrane is an assembly of ATPase molecules at the poles of the cell and that the ATPase isolated from C. jejuni is serologically and structurally unusual.
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PMID:The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni. 297 56

Although activation and conversion of the inactive/latent high-molecular-weight phosphatases to the Mr = approximately 35,000 catalytic subunits can be achieved by several procedures including freezing/thawing in 0.2 M 2-mercaptoethanol and digestion by proteolytic enzymes, strong indications have been obtained that they are distinct stimulating processes. For instance, when pig brain extracts were treated with trypsin, the type-1 phosphatase could be stimulated approximately 40 fold while the type-2 phosphatase was not slightly modified. Conversely, when the same crude extracts were treated with freezing/thawing in 2-mercaptoethanol, the type-2 phosphatase could be stimulated approximately 10 fold but surprisingly the type-1 species was not slightly influenced. It is suggested that hormonal study on the activity changes of the type-1 and type-2 phosphatase in a crude extract can be made possible by the selective using of these two triggers.
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PMID:Measurement of the potential activity of the type-1 and type-2 protein phosphatases in the crude tissue extract. 300 67

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.
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PMID:Purification and characterization of two phosphorylase phosphatases from rabbit liver. 303 14

The 64K surface protein of budded Autographa californica nuclear polyhedrosis virus (AcMNPV BV) is known to play a role in the functional entry of AcMNPV BV into Spodoptera frugiperda IPLB-SF-21 cells by adsorptive endocytosis. AcV1, a neutralizing monoclonal antibody, reacts with the 64K protein and in doing so prevents efficient entry. In this communication we report that treatment of AcMNPV BV with either trypsin or proteinase K cleaves the 64K protein into one major fragment of 34.6K and two minor fragments of 36K to 37.2K that are retained with the virus. All of the fragments are glycosylated. Protease treatment does not reduce viral infectivity, but it does result in the destruction of the AcV1-reactive epitope; thus AcV1 is not able to neutralize protease-treated AcMNPV BV. Polyclonal antiserum to BV is able to recognize both cleaved and uncleaved 64K and neutralize both protease-treated and untreated virus. Protease treatment does not diminish the sensitivity of AcMNPV BV to chloroquine, but it does cause the virus to become more susceptible to inactivation by 2-mercaptoethanol (2-ME) even though exposure to 2-ME does not result in dissociation of the fragments from the virus.
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PMID:Resistance of the 64K protein of budded Autographa californica nuclear polyhedrosis virus to functional inactivation by proteolysis. 304 20

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.
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PMID:Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity. 304 19

An aspartic proteinase associated with human erythrocyte membranes was shown to be responsible for autodegradation of the membrane proteins at pH values below 5.0. When the membrane was treated with phospholipase C (Bacillus cereus) or trypsin, and simply heated at 40 degrees C, the membrane-bound latent enzyme was activated, with this being accompanied by dissociation of the enzyme from the membrane. Divalent cations such as Ca2+ and Mg2+ had an inhibitory effect on the dissociation of the membrane-bound enzyme when preincubated with the membrane. The results indicate that the activation of the membrane-bound enzyme is due probably to perturbation of the normal membrane organization. When the purified enzyme was treated with 10mM 2-mercaptoethanol at 37 degrees C, the enzyme (79-82 kDa) was converted to a low molecular mass form with 42-47 kDa without any loss of activity. With the exception of treatments by thiol-reducing reagents, no conversion was observed by a variety of procedures such as exposure to 1 M NaCl and 0.1% sodium dodecyl sulfate, treatment with trypsin and incubation at pH 3.5 for up to 15 h, indicating that the enzyme consists of two polypeptide chains held together by disulfide bonds.
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PMID:An aspartic proteinase of erythrocyte membranes. Proposed mechanism for activation and further molecular properties. 306 Jan 44

It was found that a non-tumorigenic epithelial cell line from the liver of a Buffalo-strain rat (BRL) secreted into the culture medium various inhibitors of the growth of BRL and RSV-BRL (tumorigenic BRL transformed by infection of Rous sarcoma virus). The secreted inhibitors were classified into two types: one inhibited the growth of BRL to a greater extent than that of RSV-BRL (non-tumorigenic BRL growth inhibitor, NGI), and the other, vice versa (tumorigenic BRL growth inhibitor, TGI). Two NGI (NGI-I and NGI-II) and two TGI (TGI-I and TGI-II) were highly purified from the serum-free conditioned medium. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis without 2-mercaptoethanol, NGI-I and II gave protein bands with molecular weights (Mr) of 56,000 and 21,000, respectively. TGI-I and II gave a band that migrated faster than bromophenol blue marker dye, but they did not pass through an ultrafiltration membrane with an Mr cutoff of 5,000. In the presence of a reducing reagent, only NGI-II showed a decrease of Mr, from 21,000 to 11,000. NGI and TGI showed 50% growth inhibition with BRL and RSV-BRL, respectively, at 5-15 ng/ml in the medium containing 10% fetal calf serum. NGI and TGI all were stable to 1 M acetic acid (pH 2.3) and 6 M urea, but labile to 5 mM dithiothreitol or trypsin. Of the eight cell lines tested, NGI-I was most effective on BRL, NGI-II on BRL and HSC-3 (human tongue squamous carcinoma), and both TGI-I and II on RSV-BRL.
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PMID:Multiple forms of growth inhibitors secreted from cultured rat liver cells: purification and characterization. 317 May 13


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