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 amino-terminal sequence and composition of the subunits of the hemagglutinin (HA) of influenza virus has been determined. The hemagglutinin has been isolated by two techniques. (1) as the intact hemagglutinin after disruption of the virus in sodium dodecyl sulfate, giving 2 subunits of 58,000 daltons (HA1) and 26,000 daltons (HA2), and (2) after treatment of the virus with bromelain, giving 2 subunits of 58,000 daltons (BHA1) and 21,000 daltons (BHA2). In both preparations these subunits are linked by disulfide bonds. The aminoterminal sequences of HA1 and BHA1, and HA2 and BHA2 are the same. The composition of the 50 residue peptide associated with the membrane, which is removed from the C-terminus of HA2 by bromelain, is deduced and shown to be hydrophobic and contain 50% of the serine residues of HA2. The biosynthetic precursor of the hemagglutinin has been purified from the membranes of abortively infected chick fibroblasts and shown to have the same amino terminus as HA1. Thus the order of biosynthesis is NH2-HA1-HA2-COOH. The amino-terminal sequence of BHA2--at the cleavage site of the precursor--is shown to be a palindrome: NH2-Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-. This sequence is conserved in representative viruses from each of the major pandemics. A region of homologous sequence is described between the hemagglutinins of influenza type A and B viruses.
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PMID:Studies on the primary structure of the influenza virus hemagglutinin. 105 18

Although neutrophils are not viewed as a principal defense against influenza A virus (IAV) infection, their interactions are both complex and clinically relevant. Activation of the neutrophil is distinctive from that described for chemoattractants. To more fully characterize the pathway by which IAV stimulates the human neutrophil, we have examined its binding characteristics. First, inhibition studies with various sialic acid-containing and sialic-free sugars showed that IAV binds to sialic acid residues and activates receptors distinct from those used by Concanavalin-A (Con-A) and formyl-methionyl-leucyl-phenylalanine (FMLP) and that overlap those bound by wheat germ agglutinin (WGA). That viral hemagglutinin (HA) mediates viral binding and activation was shown by preincubating neutrophils with purified monovalent bromelain-released HA (BHA) and showing that IAV-induced membrane depolarization and hydrogen peroxide (H2O2) production were inhibited approximately 95%. However, binding inhibition required significantly higher concentrations of purified HA, suggesting that binding and cell activation have different interactive requirements. Desialation of the neutrophil surface membrane by neuraminidase treatment resulted in a 90.6% +/- 4.4% and 53.1% +/- 8.7% inhibition of IAV activation of neutrophils and viral binding, respectively. Resialation with ganglioside GT1b totally restored viral binding, but did not reverse the inhibition of activation. Thus, although HA was shown to mediate binding and neutrophil activation, viral binding per se was insufficient to stimulate the cell. Having demonstrated the functional role of HA, we sought to establish the mechanism of stimulation. HA in three different forms (BHA, HA-rosettes, and HA-liposomes) failed to activate the cell, although H2O2 production evoked by IAV stimulation was reduced in competitive inhibition studies with each preparation. Upon cross-linking with a monoclonal antibody to HA, activation comparable to that of intact virus was observed. The requirement for cross-linking of functional receptors, as opposed to activation through the neutrophil Fc receptor, was confirmed in experiments using staphylococcal A protein. These studies have shown the chemical specificity of IAV binding to the human neutrophil, the character of the receptor(s) stimulated to activate the IAV-evoked response, and the activation requirement for cross-linking those receptors responsible for stimulating functional responses.
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PMID:Influenza A virus binding to human neutrophils and cross-linking requirements for activation. 133 33

A model is proposed for the study of molecular mechanisms of a low pH-induced interaction of fusion proteins of enveloped viruses and cell membranes. The model consists of large monolamellar liposomes containing ionophore nigericin in their membranes and ectodomains of fusion protein in their inner space. The process of interaction of the protein with the lipid bilayer is triggered by acidification of the liposomal constituents to the pH of fusion with the help of nigericin by adding citric acid to the outer medium. To visualize the protein structural reorganization, the tritium planigraphy was used. Comparison of the values of specific labelling of the proteins and distribution of radioactivity in individual amino acids in control (at neutral pH) and experimental liposome samples (at the pH of fusion) permits to realise the character of protein-membrane interaction. We have obtained the first results in the study of interaction of the bromelain-released soluble ectodomain of the HAXX molecule (BHA)--with the lipid membrane. The observed increase in the protein specific activity and selective increase in the specific activity of hydrophobic amino acids Ile, Phe and Tyr in experimental liposome samples as compared with the controls did not contradict to the conventional concept, that a hydrophobic N-terminus of HA2 subunit of hemagglutinin is responsible for its interaction with lipid membranes.
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PMID:A model for the study of the mechanism of a low pH-induced interaction of the virus fusion proteins and cell membranes. 195 9

Toward elucidating molecular details of virus-induced membrane fusion, we have studied the low pH-triggered interaction of the bromelain-solubilized ectodomain of influenza hemagglutinin with liposomes. Polypeptide segments which insert into the apolar phase of the lipid bilayer were first labeled specifically using either of the two membrane-restricted carbene-generating reagents, 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine and 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] undecanoyl]-sn-glycero-3-phosphorylcholine, and were then identified on the basis of cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine-skatole fragment analysis and Edman degradations. Here, we demonstrate that the hydrophobic interaction is mediated solely by the so-called "fusion peptide" which corresponds to the NH2-terminal segment of the BHA2 subunit of nature influenza hemagglutinin. Predominant sites of labeling within that segment were Phe-3, Ile-6, Phe-9, Trp-14, Met-17, and Trp-21. The average 3-4 residue spacing between consecutive labeled amino acid side chains suggests a helical structure of that segment with an amphiphilic character.
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PMID:Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide". 270 99

New thiol protease inhibitors, estatins A and B, were isolated from the culture filtrate of Myceliophthora thermophila M4323. The basic, water-soluble inhibitors were characterized as having an agmatine, trans-epoxysuccinic acid and L-phenylalanine or L-tyrosine moieties in the structure. The molecular formulas C18H25N5O5 and C18H25N5O6 for A and B were indicated by elemental analysis and fast atom bombardment MS. Estatins were specific inhibitors against thiol proteases such as papain, ficin and bromelain. They suppressed IgE antibody production in mice, but not IgG.
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PMID:Isolation and characterization of new thiol protease inhibitors estatins A and B. 279 91

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.
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PMID:Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein. 296 15

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.
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PMID:Ananain: a novel cysteine proteinase found in pineapple stem. 319 29

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.
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PMID:L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay. 639 97

The digestion of human GH (hGH) with the proteolytic enzyme, bromelain, results in a major product consisting of a mixture of three large fragments, i.e. residues 1-135 + 143-191, 1-135 + 145-191, and 1-135 + 146-191. In the case of each fragment, the N-terminal peptide is joined to the C-terminal fragment by the disulfide bridge between residues 56 and 165. A C-terminal fragment mixture consisting of peptides 143-191, 145-191, and 146-191 was isolated from this major digestion product after reduction and S-carbamidomethylation of its disulfide bonds. In the present study, the noncovalent complementation of the peptides in this mixture with S-carbamidomethylated peptide 1-134 derived from thrombin-digested hGH was investigated. Noncovalent complementation of these peptides was accomplished by dissolving equimolar amounts of the materials in 0.5% ammonium bicarbonate-6 M guanidine-HCl and dialyzing the mixture slowly to remove the guanidine-HCl. The recombinant mixture was recovered in 26% yield by gel filtration of the peptide mixture and was found to contain three noncovalent recombinant species, i.e. peptides 1-134 + 143-191, 1-134 + 145-191, and 1-134 + 146-191. Thus it would appear that residues 135-145 are not required to obtain noncovalent complementation between the N- and C-terminal regions of the hGH molecule. In an RIA for hGH the recombinant mixture was found to possess approximately 40% the cross-reactivity of the native hormone. In contrast, it had only about 10% the activity of native hGH in the weight gain test in hypophysectomized rats, in stimulating phenylalanine incorporation into the protein of the isolated hypophysectomized rat diaphragm, and in stimulating glucose oxidation by isolated adipose tissue of hypophysectomized rats. The limited biological activity of the recombinant mixture is of interest, since the major bromelain digestion product from which the C-terminal peptides were derived consists of a mixture of rather similar molecules (i.e. peptides 1-135 + 143-191, 1-135 + 145-191, 1-135 + 146-191; and with intact disulfide bridges), which exhibits substantial growth-promoting and insulin-like activities.
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PMID:Complementation of human growth hormone (GH) peptide 1-134 with C-terminal fragments of human GH produced by digestion with bromelain. 682 9

A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. alpha-Chymotrypsin, papain, thermolysin and bromelain adsorbed on Celite were used as catalysts. Organic solvents such as acetonitrile and ethyl acetate with small amounts of buffer added or at specific water activity were used as reaction media. Simple readily available amino acid ester derivatives were used as starting building blocks. This feature allowed the possibility of using the products in one step directly as acyl-donor ester, without any chemical or enzymatic modification, in the next enzymatic coupling. The optimal strategy for the synthesis of the enkephalin derivatives was different depending on the carboxy terminal group. The preparation of the carboxy-terminal amide derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-NH2) was achieved via 4 + 1 fragment condensation catalyzed by alpha-chymotrypsin. The carboxy-terminal ethyl ester derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-OEt) were obtained via 2 + 3 condensation catalyzed by bromelain, a quite unusual protease for peptide synthesis but more effective than papain in this coupling. Both syntheses were carried out in four enzymatic steps and one or two chemical deprotection steps routinely used in peptide synthesis. The overall yields of pentapeptide derivatives were between 40-54% of pure product.
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PMID:Enzymatic peptide synthesis in low water content systems: preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives. 760 86


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