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)

A protease (melain G) was isolated from the greenish fruits of the bead tree, Melia azedarach var. japonica Makino. Melain G shares 110 identical amino acid residues (50%) with papain, 112 (51%) with actinidain, and 91 (41%) with stem bromelain. From the sites cleaved in the oxidized insulin B-chain and synthetic oligopeptide substrates by melain G, the enzyme preferred small amino acid residues such as Gly or Ser at the P2 position and negatively charged residues such as glutamic or cysteic acid at the P3 position. This is clearly different from the specificity of papain, which prefers the large hydrophobic amino acid residues such as Phe, Val, and Leu at the P2 position. Accordingly, it is presumed that the bottom of the S2 pocket of melain G is shallow due to the presence of a Phe residue, and a bulky P2 substrate (for example Phe residue) is not preferred by the enzyme. Negatively charged residues at the P3 position of substrates well suited the S3 site of melain G for making a salt bridge. It is likely that Arg61 is the S3 position of melain G by analogy with papain.
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PMID:Melain G, a cysteine protease from green fruits of the bead tree, Melia azedarach: a protease affected by specific amino acids at P3 position. 1008 36

A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography. The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits. Its molecular mass was estimated to be ca. 23 kDa by SDS-PAGE, and its sugar content was zero. Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide. The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain. The amino acid residues forming the substrate-binding the S2 pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively. As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain R than in papain.
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PMID:Amino acid sequence and some properties of phytolacain R, a cysteine protease from full-growth fruits of pokeweed, Phytolacca americana. 1039 17

A new papain-like cysteine peptidase isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.), has been purified and characterized. The enzyme, named macrodontain I, is the main proteolytic component present in fruit extracts and was purified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narrow saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The alanine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-l-amino acid p-nitrophenyl esters. The N-terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequence similarity to the other pineapple-stem-derived cysteine endopeptidases, being 85.7, 85. 2, and 77.8% identical to comosain, stem bromelain, and ananain, respectively. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.
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PMID:Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) harms (Bromeliaceae). 1068 43

A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.
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PMID:Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana. 1104 92

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.
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PMID:Characterization of a partially folded intermediate of stem bromelain at low pH. 1178 97

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.
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PMID:Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain. 1213 95

Antienzyme polyclonal antibodies against stem bromelain were raised in male albino rabbits and the Fab' monomers isolated from the IgG of the immune sera. Incubation of bromelain with the Fab' resulted in binding and gel filtration of the resulting complex suggested a 1:1 stoichiometry. Complexing with the Fab' resulted in significant stabilization of bromelain against thermal inactivation and alkaline pH.
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PMID:Binding of antibromelain monomeric Fab' improves the stability of stem bromelain against inactivation. 1263 19

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.
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PMID:Trifluoroethanol-induced "molten globule" state in stem bromelain. 1272 17

Effect of pH over a range of 0.8-10 on bovine serum fetuin (BSF) was observed by far and near-UV circular dichroism (CD) spectroscopy, intrinsic tryptophan fluorescence and ANS fluorescence measurements. It has been reported earlier by our group that a molten globule (MG) state exists in alpha-chymotrypsinogen [Biochim. Biophys Acta 1481 (2000) 229] and stem bromelain [Eur. J. Biochem. 269 (2002) 47] at low pH. In this paper we have shown the presence of an MG form of fetuin at low pH. The far-UV CD spectra showed the regain of secondary structure at pH 1.8 as compared to the complete loss of secondary structure in presence of 6 M GnHCl. Near-UV CD spectra showed disruption of tertiary structure at pH 1.8. Tryptophan fluorescence studies indicated that there is only a slight red shift in the wavelength emission maxima (lambdamax) of fetuin at low pH as compared to a significantly red-shifted spectrum of the completely unfolded state in 6 M GnHCl, indicating that the tryptophan environment of fetuin at low pH resembles more the native form. ANS binding experiments also showed an enhancement in ANS binding with decrease in pH up to 1.8. ANS binding was absent at pH 7 and in the presence of 6 M GnHCl. Fluorescence quenching experiments were also performed with acrylamide, cesium chloride and potassium iodide. The quenching of tryptophan fluorescence by the three different quenchers indicates that low pH induces a conformational change in protein, making the tryptophan residue less accessible to solvent. This suggests that a more compact structure exists at low pH. The results, being in accordance with far-UV CD and fluorescence studies, imply the presence of MG state of fetuin at low pH. As studied by fluorescence spectroscopy, denaturation of fetuin at low pH was found to be reversible.
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PMID:Characterization of molten globule state of fetuin at low pH. 1287 35

In this report, an overview is presented of studies on the three-dimensional structure in solution of glycoproteins. The investigations were carried out by using high resolution NMR spectroscopy in conjunction with molecular dynamics calculations and molecular modelling. For pine apple stem bromelain, ribonuclease 2 and the free alpha-subunit of human chorionic gonadotropin it could be shown that intramolecular interactions between carbohydrate and protein exist. Glycosylation sites are rather unique structural entities in glycoproteins, because of the local spatial arrangements. This is reflected in different intramolecular interactions between carbohydrate and protein.
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PMID:Intramolecular carbohydrate-protein interaction. 1453 95


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