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)

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.
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PMID:Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols. 2 65

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

This report examines the RP-HPLC separation of o-phthalaldehyde derivatives of amino acids, amino sugars, and amino sugar alcohols using either 2-mercaptoethanol or 3-mercaptopropionic acid. A method with pmol sensitivity for the analysis of N-acetylamino sugars of glycoconjugates was elaborated. Upon hydrolysis, amino sugars are reduced with borohydride. Automated precolumn derivatization and chromatographic conditions for the resulting hexosaminitols are the same as those used for the analysis of amino acids. The method has been tested with as little as 2 micrograms of bovine fetuin, with a glycopeptide from bromelain and with an oligosaccharide after periodate oxidation.
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PMID:Determination of amino sugars and amino acids in glycoconjugates using precolumn derivatization with o-phthalaldehyde. 138 Nov 56

Lachesis muta snake venom induced aggregation of bromelain sensitized human erythrocytes at a concentration of 1 mg/ml. The hemagglutinating protein was purified by DEAE-Sephadex A-50 column chromatography. Polyacrylamide gel electrophoresis revealed at least three bands, whereas SDS electrophoresis in the presence of 2-mercaptoethanol showed a single one. Isoelectric focusing revealed hemagglutinating activity in the range of pH 3-8. The maximum peak (mutina) at pH 5.5. This fraction was active in agglutinating human RBC of types A, B, O Rh (+) and B, O Rh (-). One mM EDTA and 1 mM Ca++ did not alter the agglutinating time significantly. Lactose and inositol inhibited the agglutination of A, B, O Rh (+) and B, O Rh (-) human RBC. The present study showed the non specificity of the hemagglutinating activity of mutina. It was also shown that mutina is a non-mitogenic protein.
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PMID:Purification and some properties of hemagglutinating protein mutina from bushmaster Lachesis muta snake venom. 367 7

In a serum free, 2-mercaptoethanol supplemented culture medium muramyl dipeptide (MDP) is able to increase the number of plaque-forming cells (PFC) directed against syngeneic, bromelain-treated red blood cells (br-MRBC) and against an autoantigen, mouse albumin. The non-specific stimulation of anti-br-MRBC PFC by MDP, as by bacterial lipopolysaccharide (LPS), can be observed in spleen cell populations depleted of adherent and phagocytic cells, and in nu/nu spleen cell cultures. However, the kinetics of the induction of anti-br-MRBC PFC in murine spleen cell cultures in presence of LPS or of MDP are not identical. Moreover, MDP is able to stimulate C3H/He Orl (LPS low-responder strain) cells. Thus, the mechanisms of non-specific stimulation by MDP or by LPS could be different. Experiments done with thirteen structural analogues of MDP showed that there exists a good correlation between the adjuvant activity and the ability to induce anti-br-MRBC PFC.
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PMID:Induction of antibodies directed against self and altered-self determinants by a synthetic adjuvant, muramyl dipeptide and some of its derivatives. 616 92

We have detected an endoglycosidase activity produced by Flavobacterium meningosepticum. This enzyme, named endo F, cleaves glycans of both the high-mannose and the complex type linked through asparagine to the protein backbone. The data indicate that cleavage occurs via hydrolysis of the glycosidic bond of the N,N'-diacetylchitobiose core structure adjacent to asparagine, similar to that due to endo H and endo D. Extreme variability was noted in the availability of this cleavage site among N-linked glycoproteins. Glycoproteins of retrovirus, lymphocytic choriomeningitis virus, Pichinde virus, and HLA-A and -B antigens were readily cleaved in the presence of nonionic detergent. Others, such as ovalbumin, fetuin, bromelain, ovomucoid, alpha 1-acid glycoprotein, immunoglobulin G, and influenza virus hemagglutinin became susceptible only after reduction and alkylation or when cleavage was performed in the presence of 1% 2-mercaptoethanol. Endo F should prove useful in the study of glycans and protein backbones as discrete entities and for defining the nature of the glycan-protein interface.
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PMID:endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. 681 50

The in vitro effect of bongkrekic acid on stem bromelain, papain and ficin was studied. The hydrolysis of casein by these enzymes was inhibited by bongkrekic acid, but the inhibition was always incomplete even with a large excess of the effector. Using a fully activated specimen of stem bromelain, purified on an organomercurial agarose affinity column, the inhibition by bongkrekic acid was not stoichiometric. The SH group of cysteine remained intact after incubation with an excess of bongkrekic acid at 24 degrees C for 20 min. However, partial inhibition of stem bromelain by bongkrekic acid was reversed by incubation at 37 degrees C for 5 min with 5mM cysteine or 2-mercaptoethanol. Ethylene glycol and glycerol had no such restorative effect. These results indicate that molecules of bongkrekic acid are non-covalently bound to a thiol protease, only partially and reversibly shielding its essential SH group.
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PMID:Effect of bongkrekic acid, a product of Pseudomonas cocovenenans, on thiol proteases. 716 5

This study was designed to describe an accurate and consistent microscopic technique for the assessment of sperm number and motility in sperm-cervical mucus samples, such as those of postcoital tests (PCTs), and to identify a suitable method to extract functional spermatozoa from cervical mucus (CM). Sperm-CM preparations containing various sperm concentrations were counted using three different microscopic illuminations. The dark field-Makler technique was compared with the more classical bright field-slide technique currently used by our clinicians. Several sperm extraction techniques were applied first to bovine (BCM) and then to human (HCM) cervical mucus. Dark field microscopic illumination provided accurate, fast, and easy sperm identification. Counting variability was significantly greater with bright field-slide than with dard field-Makler, while sperm motility was always higher with this latter methodology. A high degree of agreement (intraclass correlation coefficient = 0.965) among three raters, i.e., low interobserver variability, was obtained only with dark field-Makler. Extraction procedures based on "swim-out," Percoll, trypsin, an enzyme cocktail, and mercaptoethanol resulted in small sperm yields in BCM. Mercaptoethanol and trypsin also showed poor sperm recovery in HCM. Among the protocols with the largest yields, the mechanical technique had the largest amount of residual CM, and bromelain reduced sperm motility. The extraction with dithiothreitol (DTT) showed the best results with a mean sperm recovery of 76% and enhanced sperm motility. Sperm viability as well as spontaneous and induced acrosome reaction were conserved in all techniques. In conclusion, use of the dark field-Makler counting technique in combination with DTT extraction of spermatozoa from CM samples, such as those of PCTs, would allow accurate and functional assessment of spermatozoa for preliminary contraceptive efficacy or infertility evaluation.
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PMID:Evaluation of human spermatozoa in cervical mucus: comparison of different microscopic and extraction techniques. 1117 93

Chelation of mercuric ions by an iminodiacetate-Sepharose gel was evaluated. The retentive properties of iminodiacetate-Sepharose gel column was studied towards proteins varying the composition of eluting systems from 2-mercaptoethanol to NaCl and imidazole, determining also the extent of mercury leaching. It was demonstrated that chelated mercury contained free sites for interaction with proteins such as bromelain and recombinant human granulocyte colony stimulating factor from E. coli. The extraction of the latter by chromatography of its inclusion bodies solution on Hg(II)-loaded Sepharose-iminodiacetate gel was also evaluated.
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PMID:Chelated mercury as a ligand in immobilized metal ion affinity chromatography of proteins. 1120 30

Rhesus (Rh) antigens are carried by a membrane complex that includes Rh proteins (D and CcEe), Rh-associated glycoproteins (RhAG), and accessory chains (LW and CD47) associated by noncovalent bonds. In heterologous expression systems, RhAG and its kidney orthologs function as ammonium transporters. In red blood cells (RBCs), it is generally accepted that NH(3) permeates by membrane lipid diffusion. We have revisited these issues by studying RBC and ghosts from human and mouse genetic variants with defects of proteins that comprise the Rh complex. In both normal and mutant cells, stopped-flow analyses of intracellular pH changes in the presence of inwardly directed methylammonium (CH(3)NH(+)(3)+CH(3)NH(2)) or ammonium (NH(+)(4)+NH(3)) gradients showed a rapid alkalinization phase. Cells from human and mouse variants exhibited a decrease in their kinetic rate constants that was strictly correlated to the degree of reduction of their RhAG/Rhag expression level. Rate constants were not affected by a reduction of Rh, CD47, or LW. CH(3)NH(2)/NH(3) transport was characterized by (i) a sensitivity to mercurials that is reversible by 2-mercaptoethanol and (ii) a reduction of alkalinization rate constants after bromelain digestion, which cleaves RhAG. The results show that RhAG facilitates CH(3)NH(2)/NH(3) movement across the RBC membrane and represents a potential example of a gas channel in mammalian cells. In RBCs, RhAG may transport NH(3) to detoxifying organs, like kidney and liver, and together with nonerythroid tissue orthologs may contribute to the regulation of the systemic acid-base balance.
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PMID:Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells. 1557 41


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