Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dissociation constants for binding of sialic acid derivatives to the hemagglutinin on intact influenza virus were determined using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constants determined with whole virus are similar to, but slightly higher than, those determined with BHA (hemagglutinin released from virus by treatment with the protease bromelain; Sauter et al., 1989, Biochemistry 28, 8388-8396), indicating that the sialic acid binding site is not significantly altered when hemagglutinin is released from virus. Binding was quantified by observing the concentration-dependent broadening of the sialoside resonances in the presence of X-31 virus or alternatively by observing the effect of the sialoside on the resonances of a competitive "reporter" ligand. The glycosidic substituent attached to the sialic acid makes relatively little difference in the affinity of the sialoside for virus: alpha(2,6)-sialyllactose (KD = 2.7 mM) binds only slightly more tightly than alpha(2,3)-sialyllactose (KD = 3.5 mM). However, inversion of the glycosidic center produces a dramatic change in affinity: the dissociation constant for the alpha-methyl glycoside of sialic acid is 4.2 mM, but not binding is observed with the beta-methyl glycoside.
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PMID:Proton nuclear magnetic resonance studies of the binding of sialosides to intact influenza virus. 164 79

At the pH required to trigger the membrane fusion activity of the influenza virus haemagglutinin (HA) the soluble ectodomain of the molecule, BHA, which is released from virus by bromelain digestion, aggregates into rosettes. Analyses of soluble proteolytic fragments derived from the rosettes indicated that aggregation is mediated by association of the conserved hydrophobic amino-terminal region of BHA2, the smaller glycopolypeptide component of each BHA subunit. Further analyses of the structure of the soluble fragments and of HA in its low pH conformation by electron microscopy, spectroscopy and in crosslinking experiments showed that, although the membrane distal globular domains lose their trimer structure at the pH of fusion, the central fibrous stem of the molecule remains trimeric and assumes a more stable conformation. The increase in length of BHA2 at low pH observed microscopically appears to result from movement of the amino-terminal region to the membrane proximal end of the molecule and in virus incubated at low pH the amino terminus may insert into the virus membrane. The consequences of these possibilities for the mechanism of membrane fusion are discussed.
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PMID:Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion. 318 28

The carbohydrates of BHA, a solubilized hemagglutinin of influenza virus by bromelain digestion, were quantitatively released as oligosaccharides by hydrazinolysis. The oligosaccharide mixture was separated into a neutral and two acidic fractions by paper electrophoresis. Both acidic fractions were resistant to sialidase digestion but were slowly converted to the neutral fraction by incubation with sulfatases. The neutral fraction which comprised about 80% in molar ratio of total oligosaccharides was separated into 13 oligosaccharides by paper chromatography and by Con A-Sepharose column chromatography. Structural studies of these oligosaccharides by sequential exoglycosidase digestion and by methylation analysis revealed that BHA contains a series of high mannose type and bi-, tri-, and tetraantennary complex type sugar chains. Occurrence of Gal beta l leads to 3GlcNAc outer chain in two and bisectional N-acetylglucosamine in one of the biantennary sugar chains is an interesting characteristic of the sugar chains of BHA.
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PMID:Carbohydrates of influenza virus hemagglutinin: structures of the whole neutral sugar chains. 683 Jul 58

The site of bromelain cleavage in the haemagglutinin of the Hong Kong influenza virus A/Memphis/102/72 has been determined by using a diagonal peptide mapping procedure on the thermolytic digest of amidated BHA. The data show that bromelain cleavage removes the C-terminal 46 residues from HA2, and that the new carboxyl-terminal residue of BHA2 is Gly 175. This is close to the beginning of the hydrophobic membrane-interacting sequence that starts at residue 183.
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PMID:The location of the bromelain cleavage site in a Hong Kong influenza virus Haemagglutinin. 728 98

The tryptophan and tyrosine content of the bromelain-released subtype H3 haemagglutinin (H3 BHA) of influenza virus were measured by u.v. absorption and fluorescence techniques. The values obtained (8 and 18 residuces, respectively) are in close agreement with those derived from amino acid analysis. Essentially all of th tryptophan residues are demonstrated to be localized on the surface of the BHA molecule.
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PMID:Influenza virus haemagglutinin: estimation of tryptophan and tyrosine content and localization of tryptophan residues. 746 4

Activation of the membrane fusion potential of influenza haemagglutinin (HA) at endosomal pH requires changes in its structure. X-ray analysis of TBHA2, a proteolytic fragment of HA in the fusion pH conformation, indicates that at the pH of fusion the 'fusion peptide' is displaced by > 10 nm from its location in the native structure to the tip of an 11 nm triple-stranded coiled coil, and that the formation of this structure involves extensive re-folding or reorganization of HA. Here we examine the structure of TBHA2 with the electron microscope and compare it with the fusion pH structure of HA2 in virosomes, HA2 in aggregates formed at fusion pH by the soluble, bromelain-released ectodomain BHA and HA2 in liposomes with which BHA associates at fusion pH. We have oriented each HA2 preparation for comparison, using site-specific monoclonal antibodies. We conclude that the structural changes in membrane-anchored and soluble HA preparations at the pH of fusion appear to be the same; that in the absence of a target membrane, the 'fusion peptide' of HA in virosomes associates with the virosome membrane so that HA2 is membrane bound at both N- and C-termini, which implies that inversion of the re-folded HA can occur; and that the structural changes observed by X-ray analysis do not result from the proteolytic digestions used in the preparation of TBHA2.
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PMID:Electron microscopy of antibody complexes of influenza virus haemagglutinin in the fusion pH conformation. 783 35

An alpha-sialoside linked to acrylamide by a short connector (5-acetamido-2-O-(N-acryloyl-8-amino-5-oxaoctyl)-2,6-anhydro-3,5-d ideoxy-D-galacto-alpha-nonulopyranosonoic acid, 1) was prepared. Compound 1 formed high molecular weight copolymers with acrylamide, derivatives of acrylamide, and/or vinylpyrrolidone upon photochemically-initiated free radical polymerization. Those copolymers for which the substituents on the acrylamido nitrogen were small inhibited the agglutination of chicken erythrocytes induced by influenza virus (X-31 (H3N2); a recombinant strain of A/Aichi/2/68 (H3N2) and A/Puerto Rico/8/34 grown in chicken eggs). The inhibitory power of the polymers depended strongly on the conditions of polymerization and the sialic acid content of the polymer. The strongest inhibitors were copolymers (poly(1-co-acrylamide)) formed from mixtures of monomer containing [1]/([1] + [acrylamide]) approximately 0.2-0.7; these copolymers inhibited hemagglutination 10(4)-10(5) times more strongly than did similar concentrations of alpha-methyl sialoside (calculated on the basis of the total concentration of individual sialic acid groups in the solution, whether attached to polymer or present as monomers). Samples polymerized in the presence of low concentrations of cross-linking reagents (bis(acrylamido)methane, BIS, and 2,2'-bis(acrylamido)ethyl disulfide, BAC) also showed increased inhibition (10-10(3)-fold relative to monomers), but their use was limited by their poor solubility. Sterically demanding substituents on any position of the acrylamide component (substituents attached to the vinyl group or N-alkyl groups that are larger than hydroxyethyl) reduced the inhibitory power of the polymer. A 1H NMR assay and a fluorescence depolarization assay showed that poly(1-co-acrylamide) bound to a solubilized trimeric form of the viral receptor for sialic acid (bromelain cleaved hemagglutinin, BHA), less tightly than 1, on a per sialic acid basis. A similar result was also obtained with a model system comprising lactic dehydrogenase (a tetramer) and polymeric derivatives of oxamic acid: that is, poly((28, 29, 30, or 31)-co-acrylamide) had a higher inhibition constant for tetrameric lactic dehydrogenase than did the corresponding monomers (28, 29, 30, or 31) on a per oxamate basis. Poly(1-co-acrylamide) is, in principle, capable of inhibiting the agglutination of erythrocytes by several mechanisms: (1) entropically enhanced binding of the polymer (acting as a polyvalent inhibitor) to the surface of the virus; (2) steric interference of the approach of the virus to the surface of the erythrocyte by a water-swollen layer of the polymer on the surface of the virus; (3) aggregation of the virus induced by the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems. 793 70

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves a conformational change in the protein that leads to the insertion of a "fusion peptide" of the protein into the target membrane. It has been suggested that this insertion, aided by the formation of a complex of multiple HA trimers, would lead to perturbation of the bilayer structure of the membrane, initiating fusion. Here we present data showing that the interaction of the bromelain released ectodomain of the protein (BHA) with liposomal membranes at low pH leads to pore formation, at least at low temperatures. Strongly temperature-dependent low pH-induced inactivation of BHA resulted in a complete lack of activity of BHA above 10 degrees C. Even at 0 degrees C, only about 5% of the BHA participated in pore formation. Viral HA was less rapidly inactivated and still induced pores at 37 degrees C. BHA-induced pore formation showed a sigmoidal time course. Once BHA had formed a pore in one liposome, it did not form a pore in a further liposome. Quantitative analysis of pore formation indicated that one single BHA trimer sufficed to produce a pore. These data indicate that fusion peptide insertion perturbs the membrane and that the formation of a complex of trimers is not a prerequisite for the perturbation.
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PMID:Pores formed by influenza hemagglutinin. 937 9

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.
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PMID:pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers. 982 2

The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66-76, 1996, and J. Virol. 71:4062-4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the alpha-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.
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PMID:pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion. 997 55


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