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)

The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.
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PMID:[The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86]. 222 28

Splenic lymphocytes from BALB/c mice immunized with "cores" of influenza virus, obtained after bromelain cleavage of the surface glycoprotein, were fused with the P3-NS1/1-Ag-1 mouse cell line to yield hybridoma cultures. Among 20 stable cloned hybrid cells secreting monoclonal antibodies, one was specific for the nucleoprotein (NP), 11 were specific for the membrane (M) protein and eight were specific for the hemagglutinin (HA). These "cores" used as immunogen contained only the internal proteins of the influenza virus, namely the three polymerases, the NP and the M protein and no HA when examined by standard procedures of SDS-PAGE, electron microscopy and hemagglutination activity. It thus appeared that a small amount of contaminating antigens can sensitize a sufficient number of mouse B cells to be selected as hybrid partners. These antibodies were provisionally assigned as anti-carbohydrate attached to the HA.
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PMID:Hybridoma antibodies produced against bromelain derived cores of influenza virus. 240 34

Antibodies against horseradish peroxidase (anti-HRP) recognize neural specific cell surface antigens in Drosophila and other insects. The nature of these antigens was investigated in Drosophila and found to include a complex set of developmentally regulated proteins. Their common epitope appears to be a carbohydrate that shares features with the sugar moiety of pineapple stem bromelain, a plant glycoprotein whose carbohydrate structure has been determined. A mutation was identified that eliminates staining by the antibody in imaginal and adult neural tissue. Tissue specific glycoconjugates, although widespread in the animal kingdom, are little understood. This mutation provides a unique opportunity to address the consequences of altering a neural specific carbohydrate moiety in an otherwise intact and behaving animal. The mutation maps to 84F. A second mutation, contained on the third chromosome balancer, TM3, eliminates anti-HRP staining in embryos. These mutations appear to be separate genes.
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PMID:A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate). 246 62

Immunoglobulin fragments, whether of polyclonal or monoclonal antibodies, offer a number of advantages over the intact immunoglobulin. The generation of immunoreactive fragments from monoclonal antibodies (MAb) is not always a straightforward task. Both pepsin and papain can be used to digest MAb to a bivalent molecule with a Mr of 100,000. However, pepsin pepsin digestions does not always result in immunoreactive fragments and a stable consistent product by papain digestion is often difficult to obtain. MAb B72.3 is an example of both situations. MAb B72.3 reacts with a glycoprotein (TAG-72) with a molecular weight greater than 10(6). MAb B72.3 has been shown to exhibit a high degree of selective reactivity with colon, breast and ovarian carcinomas and has been used for radioimmunodiagnosis in model systems and in clinical trials. A third enzyme, bromelain, in the same family of sulfhydryl proteases as papain, has been used to generate a fragment of MAb B72.3, with a Mr of approximately 100,000. The bromelain-generated fragment of MAb B72.3 retained 100% immunoreactivity as measured in competitive solid-phase radioimmunoassays and could be generated with consistent results from one preparation to another. Both the bromelain- and papain-generated fragments were radiolabelled with 125I without significant loss of the MAb's reactivity to tumor extracts. Differences were observed between the bromelain- and papain-generated fragment when compared in vivo. Fragmentation of MAb B72.3 with bromelain has yielded a superior bivalent fragment for radioimmunolocalization.
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PMID:Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas. 249 38

The Minnesota strain of turkey enteric coronavirus (TCV) was grown on a human rectal tumor (HRT-18) cell line in the presence of radiolabeled amino acids and glucosamine to analyse virion structural proteins. In addition to the 52,000 unglycosylated nucleocapsid protein, three major glycoprotein species were found to be associated with the viral envelope. A predominant glycosylated protein with a molecular weight of 22-24,000 represented the transmembrane matrix protein. Larger glycoproteins with apparent molecular weights of 180-200,000 (gp 200), 120-125,000 (gp 120) and 95-100,000 (gp 100) were associated to the characteristic large bulbous projections (peplomers) located at the surface of the virion. The gp 100 and gp 120 species apparently arose from a proteolytic cleavage of gp 200, as suggested by digestion studies with trypsin and chymotrypsin. An additional large glycoprotein with mol. wt. of 140,000 (gp 140), that behaved as a disulfide-linked dimer of a 66,000 molecule, was found to be associated to granular projections located near the base of the large peplomers. Digestion studies with trypsin, bromelain and pronase demonstrated that gp 140 was related to the hemagglutinating activity of the virus. An inner membranous sac or tongue-shaped structure could be visualized in the interior of the viral particles following treatment with pronase. In contrast, trypsin or chymotrypsin treatments resulted in evaginations ("budding") on the virus surface. Progeny viral particles produced in TCV-infected cell cultures in the presence of tunicamycin lacked both types of surface projections, as demonstrated by electron microscopy and electrophoresis. The matrix protein also appeared to be reduced to its unglycosylated form, concomitant with a considerable loss of its antigenicity. Thus, with respect to its morphological and biochemical characteristics, TCV resembles viruses belonging to the group of mammalian hemagglutinating coronaviruses, but differs in that both types of envelope glycoproteins are N-glycosylated as in case of the avian infectious bronchitis virus.
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PMID:Identification and location of the structural glycoproteins of a tissue culture-adapted turkey enteric coronavirus. 267 55

The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus.
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PMID:Variant influenza virus hemagglutinin that induces fusion at elevated pH. 300 92

Lymphoproliferative disease virus of turkeys (LPDV), a C-type retrovirus, was shown to contain 3 major [32 kilodaltons (kd, p 32), 26 kd, 22/21 kd] and 2 minor (41 kd and 12 kd) polypeptides. Preliminary evidence suggests a glycoprotein of 76 kd (GP 76) and a major doublet polypeptide of 13.5/13 kd to be also of viral origin. Of these GP 76 was susceptible to bromelain action implying its surface location in the virion, while p 32, p 26 and p 13.5/13 were the main constituents of viral cores. p 13.5/13 bound an RNA probe, suggesting it to be the main constituent of viral ribonucleoprotein. p 22/21 was not cleaved by bromelain, and was absent in viral cores suggesting its intramembrane location between virion envelope and core. The polypeptide profile of LPDV is distinct from those of avian sarcoma-leukosis viruses and avian reticuloendotheliosis viruses.
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PMID:Characterisation of lymphoproliferative disease virus of turkeys. Structural polypeptides of the C-type particles. 303 51

Binding experiments with radioactively labelled influenza C virions were carried out to investigate the interaction of the virus with human erythrocytes. The erythrocytes from any of 35 different individuals were found to contain influenza C virus-binding sites though their number was variable among the individuals and was much less than that on mouse, rat and chicken erythrocytes. Attachment of influenza C virus to human erythrocytes was inhibited completely by prior treatment of the virus with anti-HE monoclonal antibody having a strong haemagglutination inhibition activity. Pretreatment of erythrocytes with neuraminidase or the neuraminate-O-acetylesterase of influenza C virus resulted in a marked reduction in the level of virus binding. Thus it appears that human erythrocytes have a low level of O-acetylated sialic acid-containing glycoconjugates that can interact specifically with the HE glycoprotein of influenza C virus. Proteolytic digestion of erythrocytes with ficin, bromelain or V-8 protease inhibited virus binding almost completely, suggesting that the erythrocyte receptor for influenza C virus is a glycoprotein. In contrast to these enzymes, trypsin treatment of erythrocytes reduced virus binding by only about 50%, and alpha-chymotrypsin treatment did not inhibit at all. It was also found that treatment of erythrocytes with monoclonal antibody to the M or N blood group antigen greatly inhibited virus binding to the cells. These results, taken together, suggest that most influenza C virus receptors on human erythrocytes, if not all, reside on glycophorin A which is known to possess the M or N blood group activity.
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PMID:Attachment of influenza C virus to human erythrocytes. 304 38

The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.
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PMID:Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion. 341 82

The hemagglutinin (HA) spike glycoprotein of influenza virus catalyzes a low pH-induced membrane fusion event which releases the viral genome into the host cell cytoplasm. To study the fusion mechanism in more detail, we have prepared the ectodomain of HA in water-soluble form by treating virus particles with bromelain. Under mildly acidic conditions (pH less than or equal to 5.8), the ectodomain undergoes a conformational change which we found to be biochemically and immunologically equivalent to that in native viral HA. It became sensitive to proteinase K, it exposed new antigenic epitopes in its HA1 chain, and it acquired amphiphilic properties, notably the ability to bind to liposomes. The attachment to liposomes exhibited the same pH dependence and rapid kinetics as the conformational change and was mediated by HA2. The nature of the attachment resembled that of an integral membrane protein except that the bound HA was partially removed by base. As observed for virus fusion, attachment is independent of divalent cations and lipid composition. Temperature was found to be a critical parameter only with dimyristoylphosphatidycholine vesicles where attachment was partially blocked below the major phase transition. These and other results obtained indicated that the low pH-induced conformational change in the isolated ectodomain is equivalent to that occurring in intact viral HA, and that its attachment to liposomes can serve as a model for the initial stages in the HA-induced membrane fusion reaction.
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PMID:Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. 397 12


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