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:3.4.22.32 (bromelain)
1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two small plaque mutants designated as 1a and 2c were isolated from DBT cells persistently infected with the JHM strain of mouse hepatitis virus. Unlike the wild type JHM, these two mutant viruses grew more slowly with no prominent cell fusion. The buoyant densities of the mutants were slightly lower and 2c was revealed to have fewer peplomers than JHM by electron microscopy. The purified JHM contained five polypeptides with molecular weights (M.W.) of 260,000, 105,000 (GP105), 65,000, 60,000 (P60), and 23,000 (GP23). In addition to two polypeptides, P60 and GP23, which were common to JHM and the mutants, 1a was found to contain three other specific polypeptides with M.W. of 180,000 (GP180), 110,000, and 95,000 (GP95), while 2c had GP180, GP105, GP95, and one with a M.W. of 175,000. All of these polypeptides were shown to be glycosylated except for P60. After bromelain treatment, all these viruses lost the peplomers and contained P60 and another new 18,000 dalton polypeptide.
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PMID:Characterization of small plaque mutants of mouse hepatitis virus, JHM strain. 631 77

A polypeptide proteinase inhibitor from human articular cartilage has been purified to homogeneity by stepwise Sephadex G-75, heparin-Sepharose and octyl-Sepharose affinity chromatography. The inhibitor is strongly cationic (pI greater than or equal to 10.5) and consists of two non-identical polypeptides associated by means of electrostatic and/or hydrophobic interactions. Amino acid analysis of the aggregate confirmed that the polypeptide was rich in basic, and hydrophobic amino acids and contained only one disulphide bridge. Sedimentation equilibrium studies showed that the aggregate had MW congruent to 7000 which could be dissociated into two polypeptides each of MW congruent to 3500. While the subunits were primarily serine proteinase inhibitors the aggregate form could also inhibit bacterial collagenase and pepsin but not thermolysin nor the cysteine proteinases, ficin or bromelain. Binding of 125I-labelled human cartilage inhibitor to heparin, keratan sulphate and proteoglycan subunit was demonstrated using gel exclusion chromatography but no interaction was detected with chondroitin 6-sulphate or hyaluronic acid. Binding of cartilage inhibitor subunits to link proteins was also shown by polyacrylamide electrophoresis. These data suggest that the human cartilage inhibitor may be localised at specific sites on the proteoglycan complex where it would be ideally placed to attenuate degradation by matrix proteinases or constitute part of an enzyme-inhibitor complex.
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PMID:Polypeptide proteinase inhibitor from human articular cartilage. 638 79

Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.
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PMID:Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor. 676 24

The complete amino acid sequences of the haemagglutinins (HAs) of one strain of each of the Hav1 and H2 subtypes and of three strains of the H3 subtype of influenza virus have been established. A large external fragment (BHA) of HA can be released from virus particles using the protease bromelain that cleaves the protein close to its carboxyl terminus, which is inserted in the virus membrane (see Fig 1). Here we show that a single disulphide bond joins the two component polypeptide chains BHA1 and BHA2 and establish the location of five intrachain disulphides. These disulphide bonds have been located in the three-dimensional structure of HA which is known to 3 A resolution.
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PMID:Disulphide bonds of haemagglutinin of Asian influenza virus. 746 12

The conformational behavior of the N-glycan Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta of stem bromelain as part of the intact glycoprotein was investigated and compared with that of the same N-glycan as part of a bromelain-derived glycopeptide. Proton chemical shifts of the glycoprotein N-glycan were determined by 2D HOHAHA and 2D NOESY measurements, making use of the glycopeptide 1H NMR data. During each 2D NMR experiment about 4% of the glycoprotein denatured. Experimental data concerning interproton distances of the intact glycoprotein N-glycan were obtained by NOESY 1H NMR spectroscopy. Several theoretical models for the N-glycan, obtained by molecular dynamics simulations of the glycopeptide, were investigated. Comparison of experimental and theoretical NOESY cross peak intensities was performed with the program CROSREL. In comparison with the glycopeptide, the distribution of populations between two main conformations of the Fuc alpha 1-3GlcNAc linkage was altered. In addition, the omega = 60 degrees (gt) rotamer of the Man alpha 1-6Man linkage seems to be present for a significant period of time, whereas in the glycopeptide the omega = -60 degrees (gg) conformation exists exclusively. Except for the Xyl beta 1-2Man linkage, the mobilities around the glycosidic linkages in the glycoprotein were reduced compared with those in the glycopeptide, especially concerning the Fuc alpha 1-3GlcNAc and Man alpha 1-6Man linkages. These findings might be the result of an interaction of the polypeptide chain with the Fuc alpha/Man alpha side of the N-glycan. A qualitative analysis of the NMR spectra showed a larger degree of mobility in the denatured glycoprotein N-glycan than in the intact glycoprotein.
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PMID:Conformational analysis of the xylose-containing N-glycan of pineapple stem bromelain as part of the intact glycoprotein. 779 34

NZB is a mouse strain that spontaneously develops autoimmune haemolytic anaemia at 10-12 months of age. We analysed the autoantibodies present throughout their life and compared them to natural autoantibodies found in the normal mouse. Sera and Coombs' antibodies eluted from red blood cells (RBC) were tested for their activities against RBC and a panel of antigens: actin, myoglobin, myosin, tubulin, spectrin, DNA and trinitrophenyl bovine serum albumin (TNP-BSA), F(ab')2 and Fc fragments of IgG by using enzyme immunoassays (EIA) and Western blotting analysis of RBC membrane extracts. In NZB mouse sera, activities of IgM and IgG against the whole panel, compared to those of sera from age-matched BALB/c mice, increased progressively throughout life with oscillating values in parallel with the anti-RBC activity. Two periods of autoantibody production seem to exist: the first is characterized by a fluctuating high level of IgM and stable level of IgG natural autoantibodies, and the second by a rise of IgG natural autoantibodies in parallel with IgG anti-RBC antibodies. The presence of idiotype D23 (IdD23), which is characteristic of natural polyspecific autoantibodies, was high on serum IgM and low on IgG autoantibodies throughout life. To further analyse autoantibody level oscillations, we tested IgM and IgG fractions after their separation from whole serum and observed highly enhanced autoantibody activities of both IgM and IgG. These autoreactivities markedly diminished when the separated IgM and IgG fractions were recombined, suggesting humoral control of the autoreactivity as we had already noted for IgG in normal animals. During the first period of autoantibody production, IgM and IgG antibodies eluted from RBC (Combs' antibodies) and those eluted from serum using an RBC-immunoadsorbent (circulating antibodies) reacted with all RBC membrane components, with all antigens of the panel and with F(ab')2 and Fc. Some of these reactivities were comparable to those exhibited by a monoclonal antibody recognizing bromelain-treated RBC. In the second period, both IgM and IgG Coombs' antibodies reacted more strongly with spectrin, and exhibited new specificities, for example against the band 3 polypeptide. IdD23 was abundant on Combs' IgG antibodies in the second period. Taken together, these data suggest that IgM and IgG natural autoantibodies, able to recognize not only RBC antigens but also other antigens, particularly F(ab')2 and Fc fragment of IgG, predominate in Coomb's antibody population.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Natural autoantibodies are involved in the haemolytic anaemia of NZB mice. 798 Aug 46

We have used rabbit polyclonal antisera raised against synthetic peptides complementary to different domains of the Rh polypeptides and Rh glycoprotein to examine the topography and organization of these proteins in the human erythrocyte membrane. Previously unrecognized exofacial protease sites have been identified on Rh CcEe, D proteins, and Rh glycoprotein. The Rh D protein has two specific bromelain cleavage sites located within the first and sixth predicted external domains, with the site of cleavage localized in the sixth domain to lie between residues 353 and 354. All Rh polypeptide species were found to be susceptible to cleavage with trypsin and subtilisin within the first external domain of these proteins. The Rh glycoprotein has two bromelain cleavage sites within the first external domain. These flank the single N-glycosylation site (Asn37), with the cleavage site toward the C-terminal side of this residue being between residues 39 and 40. Bromelain treatment was found to deglycosylate the Rh glycoprotein. Immunoprecipitation experiments have revealed that anti-C, -c,E, -e, and -D immune complexes are reactive with antisera raised against the fourth predicted external loop of the Rh proteins and the C-terminal domain. These data indicate that the hypothesis that suggests Rh C/c antigens are expressed on truncated Rh polypeptides by a mechanism of alternate splicing is incorrect and support the hypothesis that Rh Cc and Ee antigens are expressed on a single polypeptide chain.
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PMID:Immunochemical analysis of the human erythrocyte Rh polypeptides. 866 3

1. Hydrophobicity analysis of the monocarboxylate/proton cotransporter MCT1 (lactate transporter) suggests a structure with 12 transmembrane (TM) segments, presumed to be alpha-helical. 2. A series of anti-peptide antibodies have been raised against regions of the MCT1 sequence, which each recognize a polypeptide of approx. 40 kDa in rat erythrocytes. The topology of rat MCT1 was investigated by studying the immunoreactive fragments derived from proteolytic digestion of the protein in intact rat erythrocytes and leaky membranes. 3. Reactivity with an anti-(C-terminus) antibody was prevented on treatment of leaky membranes, but not intact cells, with carboxypeptidase Y, indicating that the C-terminus of the protein is cytoplasmically disposed. 4. Treatment of intact cells in saline buffer with trypsin, chymotrypsin, bromelain and protease K (up to 1 mg/ml) resulted in no degradation of MCT1, indicating the absence of any large exposed extracellular loop. In a buffer of low ionic strength (containing sucrose), cleavage was observed with bromelain at an extracellular site, probably TM9/10.5. Treatment of leaky membranes with low (less than 100 micrograms/ml) concentrations of several proteases resulted in fragmentation of MCT1, reflecting cleavage at the cytoplasmic face of the membrane. These treatments generated N-terminal fragments of apparent molecular mass approx. 17-19 kDa that were resistant to further degradation. The epitopes for the TM6/7 and C-terminal antibodies were either lost from the membrane or destroyed under most of these conditions, indicating that these regions of the protein are located in the cytoplasm. 6. More detailed structural prediction analysis of MCT-related sequences was made assuming the constraints placed upon the possible arrangements by the experimental data outlined above. This analysis provided additional strong evidence for the 12-TM-segment model, with cytoplasmic N- and C-terminal ends and a large internal loop between TM6 and TM7. The predicted helices were assigned moments of hydrophobicity and residue substitution; for a number of TM segments this permitted the prediction of the sides of the helix that faced membrane lipid and the interior of the protein.
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PMID:Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1). 900 67

An antigen complex unique for porcine gamma/delta T cells has previously been identified using the monoclonal antibodies MAC319 and MAC320. Here we use digestion with the proteolytic enzyme bromelain to selectively release the MAC319 antigen as a soluble fragment, for further characterisation. A cytofluorometric inhibition assay was developed to follow the purification of this fragment, as the conformation sensitivity of the MAC319 epitope prevented the use of immunoblotting techniques. The antigen has been purified using a combination of anion-exchange and hydrophobic-interaction columns, followed by separation on a size-exclusion column. Fractions from the size-exclusion column containing the antigen consisted of one major band at Mr 95,000. This species was shown to be specifically absorbed onto MAC319-coupled Sepharose, thereby identifying the MAC319 antigen. N-terminal amino acid sequencing of this band has revealed a previously unidentified sequence. This fragment was also shown to be glycosylated, most likely with a single sugar moiety. Enzymatic removal of the sugars showed that they did not appear to be necessary for binding of the polypeptide to the MAC319 antibody.
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PMID:Isolation and N-terminal sequence determination of a novel gamma/delta T cell surface antigen. 939 61

Three protein inhibitors of proteolytic enzymes with molecular weights 21, 22, and 23 kD were isolated from potato tubers (Solanum tuberosum L.) by ammonium sulfate precipitation followed by gel and ion-exchange chromatography. The 21- and 22-kD proteins were shown to be serine proteinase inhibitors with different specificities. The 21-kD protein inhibits human leucocyte elastase and trypsin effectively, but it is less effective towards chymotrypsin. The 22-kD protein is an inhibitor of cysteine proteinases and suppresses the activities of papain, ficin, and bromelain with the same affinities. None of the isolated proteins inhibit subtilisin, pepsin, or cathepsin D. The 21-kD protein consists of two disulfide-linked polypeptide chains with molecular weights of 16.5 +/- 1 kD and 4.5 +/- 1 kD. The 22-kD and 23-kD proteins have a single polypeptide chain. The N-terminal 22-25 amino acid sequences of these three proteins were determined. These sequences have significant homology to other plant inhibitors from the Kunitz soybean inhibitor superfamily.
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PMID:Potato tuber protein proteinase inhibitors belonging to the Kunitz soybean inhibitor family. 948 70


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