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)

Pineapple stem bromelain was photooxidized in the presence of Methylene Blue used as a sensitizer. The essential sulfhydryl group of the enzyme protein rapidly became inaccessible to react with 5,5'-dithiobis(nitrobenzoic acid), but the reactivity was readily regained to the original level upon treatment with dithiothreitol. Even after such reduction, the photooxidized enzyme showed a markedly decreased hydrolytic activity on casein. Spectral examination revealed that the oxidized enzyme had tyrosine residues intact. Amino acid analysis showed significant decreases in histidine, ethionine, and tryptophan residues. Photoinactivation occurred in a similar manner also in the presence of tetrathionate which reversibly blocked the essential sulfhydryl group. It is concluded that the irreversible photoinactivation of stem bromelain must be related to the oxidation of histidine, methionine, and tryptophan residues. When the photooxidation was carried out a different pH values ranging from 4.0 to 8.3, the inactivation and the decrease in histidine content were found to be markedly pH dependent. Thus, the photooxidation experiment provided a method for directly measuring the apparent pKa of the ionization of the single histidine residue in stem bromelain. Apparent pKa values of 6.4 and 7.1 were obtained for the histidine imidazole in the absence and in presence of tetrathionate, respectively. In view of these normal pKa values for an imidazole, a mechanism of ionization of the active-site group in a plant thiol proteinase is proposed, in which the validity of mechanism involving a close electronic interaction between histidine and cysteine residues is seriously questioned.
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PMID:Photosensitized inactivation of stem bromelain. Oxidation of histidine, methionine, and tryptophan residues. 112 Jan

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

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

[35S]methionine-labelled avian infectious bronchitis virus (IBV) (strain 41) and its purified protein components and virions of IBV-Beaudette were incubated with 10 proteases. Several proteases hydrolysed all or some of the membrane glycopolypeptide (M; Mr 30K) and removed about 1.3K of peptide from the amino-(N-)-terminus plus both glycans, as determined by SDS-polyacrylamide gel electrophoresis. N-terminal analysis of [3H]isoleucine-labelled M after hydrolysis by bromelain revealed that the first nine residues had been removed. After the virions had been permeabilised with saponin, a further 2.5K decrease in molecular weight was produced and this was shown to be from the carboxy-(C-)terminus. When considered with the hydropathicity plot analysis of the amino acid sequence of M (Boursnell, M.E.G. et al., 1984, Virus Res. 1, 303-313) these results suggest that as few as 9-20 N-terminal amino acid residues may protrude at the outer membrane surface and that there is a highly protease sensitive sequence of an estimated 20-25 residues at the C-terminus of M exposed in the lumen of the virion. S2 but not S1 was cleaved to a major glycopolypeptide of approximately 71K by several proteases, and to 76K by trypsin. N-terminal sequencing of the 71K glycopolypeptide revealed that it had the same N-terminus as intact S2. After hydrolysis in the presence and absence of saponin it was concluded that S2 is very sensitive to hydrolysis near its carboxy terminus at residues close to the outer membrane surface.
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PMID:Coronavirus IBV glycopolypeptides: locational studies using proteases and saponin, a membrane permeabilizer. 301 May 96

Attachment of [35S]methionine-labelled mammalian type 3 reovirus to murine L cells and human HeLa cells was studied under equilibrium conditions. Cellular attachment sites could be completely saturated with 35S-labelled reovirus, indicating that specific attachment sites for reovirus are present on the surface of these cells. We calculated that L cells possess about 86000-105000 attachment sites per cell while HeLa cells possess about 126000-147000 sites per cell for type 3 reovirus. Unlabelled reovirus was highly efficient in competing for attachment by 35S-labelled reovirus to the saturable attachment sites of both L and HeLa cells, further indicating the specificity of the interaction. We also found that unlabelled reovirus competed equally well for both binding and internalization of 35S-labelled reovirus into murine L cells, suggesting that the L cell attachment site may serve as a virus entry site. Phospholipase digestion of L cells had no effect on subsequent reovirus attachment, while treatment of L cells with moderate concentrations of bromelain (but not trypsin, proteinase K or pronase) and Vibrio cholerae neuraminidase reproducibly decreased subsequent reovirus attachment. These results and those of others (Epstein et al., 1984, Virology 133, 46-55) suggest that mammalian reoviruses attach to specific cell surface receptors on at least two species of mammalian cells to initiate the infectious cycle.
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PMID:Saturable attachment sites for type 3 mammalian reovirus on murine L cells and human HeLa cells. 639 64

SDS-PAGE and immunoprecipitation analyses were carried out on the virion and cell-associated proteins of Hantaan virus, the causative agent of haemorrhagic fever with renal syndrome (HFRS). Purified virions have a density of 1.17 g/ml in sucrose, and contain four proteins with molecular weights of 45 000 (45K), 56K, 72K and 200K, confirming recent evidence that the virus is a member of the family Bunyaviridae. Detergent treatment of virions indicates that the 45K protein is the virus nucleoprotein. Both the 72K and the 56K proteins were labelled with [3H]glucosamine and were removed from virions by bromelain treatment, indicating that they are envelope glycoproteins. The 200K protein was found only in [35S]methionine-labelled preparations. By analogy to prototype viruses of the family Bunyaviridae, these proteins were designated N, G1, G2, and L respectively. Three virus-specific proteins (N, G1, G2) were detected in virus-infected cells. These proteins were precipitable by human convalescent serum and by serum of a Rattus norvegicus trapped in the United States. No additional virus proteins were detected in infected cells. These results confirm recent morphological and RNA studies that Hantaan virus is a member of the family Bunyaviridae. Our results also support the suggestion that Hantaan virus be placed in a new genus of Bunyaviridae.
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PMID:Hantaan virus: identification of virion proteins. 654 Feb 94

The techniques for measurement of biosynthetic rates and intracellular transit times of an integral membrane protein isoenzyme have now been validated. Thus, induction of placental alkaline phosphatase in cultured HeLa cells by prednisolone and by butyrate is shown to result in its increased biosynthesis as measured by uptake of [35S]methionine into immunoprecipitated cell-surface placental alkaline phosphatase. The cell-surface placental alkaline phosphatase is liberated from the cells by proteolytic cleavage by bromelain, which results in a decrease of the placental alkaline phosphatase subunit mass from 64,000 to 62,000 daltons. The time of transit of new placental alkaline phosphatase molecules from their ribosomal site of synthesis to their terminal cell-surface, bromelain-sensitive site is approximately 55 min. This system may be useful in studies of regulation of intracellular protein processing and transport to the cell surface of proteins destined to become integral membrane proteins.
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PMID:Measurement of biosynthetic rates and intracellular transit times for a cell-surface membrane glycoprotein, alkaline phosphatase in HeLa cells. 685 22

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

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.
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PMID:Assaying for structural variation in the parvovirus capsid and its role in infection. 977 Apr 25

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


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