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)

Ca(2+)-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or alpha(1)-casein were hydrolysed with a maximum rate at 30 degrees C, pH7.5, and with 5mm-CaCl(2), but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, alpha-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150-2158]. The Ca(2+)-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca(2+)-activated neutral proteinase.
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PMID:Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle. 53 1

Rat monoclonal antibodies (mAb) against isolated pig Null T cells were derived using a novel two-colour cytofluorometric assay. One (MAC320) identified all blood CD2-sIg- 'Null' cells (present at up to approximately 6 x 10(6)/ml). Another type (MAC319 and MAC318) identified a subset comprising approximately 60% or approximately 30% of the Null cell population. This percentage appears genetically determined. This subset partially overlapped with a gamma delta T-cell receptor+ (TcR+) population which consisted of approximately 40% of Null T cells. The antibodies did not react with other leucocyte or lymphocyte populations. In non-reducing conditions, MAC320 precipitated two molecules at approximately 270,000-280,000 MW in SDS-PAGE; the larger of which was also precipitated by MAC319 (and MAC318, which binds to the same epitope). Under reducing conditions, MAC320 immunoprecipitated two or three polypeptide chains at approximately 130,000-160,000 MW; MAC319 precipitated only the largest of these polypeptides. The large MAC319+ MAC320+ molecule on one subset is removed by bromelain treatment; the smaller MAC319- MAC320+ molecule on the remaining Null cells is not bromelain sensitive. Several properties of this new antigen complex specific to pig Null T cells show that it is distinct from the ruminant T19 complex.
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PMID:Subsets of null and gamma delta T-cell receptor+ T lymphocytes in the blood of young pigs identified by specific monoclonal antibodies. 135 15

Close to 15% of the karatasin proteinase activity in the fruit juice of Bromelia plumieri (karatas) is present outside dialysis Visking tubing in 7 days in 0.2 M acetate buffer (pH) 3.5 or 6.5) containing phenyl mercuric acetate. The small proteinase(s), distinct from the 85% activity in juice due to nondialysable karatasin with a reported Mr of 24,868, separates across Spectrapore (13 kDa) membranes but not across Spectrapore with 3.5 kDa average pore diameter. The dialyzed proteinase is named karatasin-D (K-D). Purified non-Dialysable karatasin can be dissociated to what seems to be K-D by incubation in a buffer solution, containing SDS and 2-mercaptoethanol with phenyl mercuric acetate, in dialysis experiments for 8 days at room temperature using Spectrapore 13 kDa tubing. Thus, native karatasin in B. plumieri fruit juice seem to be the result of association of 2 small molecular mass K-D subunits, linked together by disulfide bonds and electrostatic forces, in equilibrium with small amounts of free K-D molecules. The amino acid composition and partial sequence of karatasin up to the 14th position from the amino terminus have discrete analogies with papain and with stem bromelain.
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PMID:Subunit structure of karatasin, the proteinase isolated from Bromelia plumieri (karatas). 136 18

Carbonic anhydrase (CA) from mouse erythrocyte membranes is recognised as an autoantigen in Western blotting experiments with FUB 1, a murine IgM monoclonal antibody that binds both phosphatidylcholine and bromelain-treated mouse red blood cells (BrMRBC). Serum from mice stimulated with lipopolysaccharide (LPS-serum) also recognises CA. From SDS-PAGE, and blotting experiments with whole mouse erythrocytes, we found two closely spaced glycoprotein bands in the 30 kD region that reacted with both FUB 1 and LPS-serum. One of the molecular weight markers, bovine carbonic anhydrase which is of a molecular weight of about 30 kD, electrophoresed in the same 30 kD region also reacted with these antibodies. Carbonic anhydrases from a range of mammalian species were found to be crossreactive with FUB 1 and LPS-serum by Western blotting, whereas human glycophorin A and human asialoglycophorin were not recognised by the antibodies. FUB 1 specifically recognises both native and denatured bovine carbonic anhydrase in ELISA assays. The serological identity of the determinants of CA and BrMRBC was confirmed by specific absorption of both FUB 1 and LPS-serum with BrMRBC and normal mouse erythrocytes. We propose that a native autoantigenic epitope on erythrocytes may be revealed by the proteolytic action of bromelain and that this determinant is associated, at least in part, with carbonic anhydrase.
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PMID:IgM natural autoantibodies against bromelain-treated mouse red blood cells recognise carbonic anhydrase. 172 1

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

A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methyl-papain-Sepharose affinity chromatography. The molecular weight, estimated by SDS-gel electrophoresis, is about 100,000 and a species of about 75,000 is formed after incubation with horse urinary kallikrein. After incubation with trypsin, only traces of biological activity were detected in tests on guinea pig ileum. The purified protein inhibits papain and bromelain, does not correct the clotting time of a kininogen-depleted human plasma, and does not affect the clotting time of plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was found in this nonpoisonous snake. The dissociation constant (Ki) for the papain-inhibitor complex is approximately 1.6 nM.
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PMID:A Bothrops jararaca plasma cysteine-proteinase inhibitor related to mammalian kininogen. 263 47

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

Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules. This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C, which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty-acid--PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an O-ester linkage.
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PMID:Attachment of human placental-type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes. 312 11

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


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