Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A hepatic bumetanide-binding protein of molecular mass 60 kDa was isolated from rat liver sinusoidal plasma membranes after photoaffinity labelling with [3H]bumetanide. The protein was purified by non-equilibrium pH gel electrophoresis/two-dimensional gel electrophoresis. The amino acid sequences of two internal fragments share 67% and 89% similarity with rat liver catalase, which has a molecular mass of 59.758 kDa. With H2O2 as a substrate, the catalytic activity was measured in rat liver plasma membrane preparations. This activity was blocked by bumetanide and aminobumetanide. Polyclonal antibodies were raised against the purified 60-kDa membrane bumetanide-binding protein. The antibody anti-Bum-Ab 60 immunoprecipitated a 60-kDa protein from rat hepatocytes. Immunoblot analysis of SDS/PAGE and two-dimensional PAGE gels confirmed that the antibody was specific for the 60-kDa bumetanide-binding protein and cross-reacted with commercially available purified bovine liver catalase. Immunofluorescence showed the presence of the 60-kDa antigen in the plasma membrane of intact hepatocytes. Western-blot analysis revealed that the protein was present in rat kidney cortex homogenate but was lacking in hepatoma cells AS-30 D, Reuber H35 FAO and HPCT cells (clone 1E3), in spleen, and in ileum. These results indicate that a plasma-membrane-derived catalase binds bumetanide in rat liver.
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PMID:The 60-kDa bumetanide-binding protein from rat liver membranes is a catalase. 770 68

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.
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PMID:Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells. 775 Jul 90

Streptomyces coelicolor ATCC 10147 produced catalases whose electrophoretic mobility varied depending on the growth phase in liquid culture. Polyacrylamide gel electrophoresis of cell extracts resulted in six catalase activity bands, which were designated Cat1 to Ca6. Of these, Cat4 appeared during all growth phases, whereas Cat1 appeared only during the stationary phase. Catalase-deficient mutants were screened by the H2O2 bubbling test following NTG mutagenesis. In all the non-bubbling mutants tested, the Cat4 activity band significantly decreased or disappeared, suggesting that Cat4 is the major catalase. Cat4 was purified to electrophoretic homogeneity and some of its properties analysed. The enzyme has a native molecular mass of 225 kDa, as determined by gel permeation column chromatography, and consists of four identical subunits of 57 kDa, as determined by SDS-PAGE. The enzyme contains 2.6 molecules of protohaem IX per tetramer, as indicated by the absorption spectrum. It was not reducible by sodium dithionite and exhibited no peroxidase activity with o-dianisidine as the substrate. All these characteristics, as well as inhibitor studies, indicate that the major vegetative catalase in S. coelicolor, unlike E. coli vegetative catalase, is a member of the typical monofunctional catalases found in eukaryotes and some bacteria.
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PMID:Characterization of the major catalase from Streptomyces coelicolor ATCC 10147. 788 56

Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.
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PMID:Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation. 788 93

We have shown that hyperoxic exposure of immature rats induces airway smooth muscle layer thickening and cell turnover parallel to that found in the airways of patients with bronchopulmonary dysplasia and chronic, severe asthma. We hypothesized that reactive oxygen species could promote the observed airway remodeling by directly stimulating signal transduction pathways that regulate cell growth. To test this hypothesis in cultured cells, we assessed the effects of hydrogen peroxide (H2O2) on mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes. The MAP kinases are a family of 40 to 46 kD cytosolic serine/threonine kinases that participate in the transduction of mitogenic signals to the cell nucleus. Quiescent cells were exposed to H2O2 (25 to 200 microns; 2 to 60 min), after which SDS-PAGE of cell extracts was performed. Western analysis using an anti-MAP kinase antiserum revealed a decrease in the mobility of the 42 and 44 kD MAP kinase bands after H2O2 exposures of 5 to 30 min, reflecting the phosphorylation at threonine and tyrosine residues required for enzymatic activity. MAP kinase activation was demonstrated by kinase renaturation assays, which showed an almost 4-fold increase in 42 and 44 kD MAP kinase activity. Down-regulation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) partially reduced H2O2-stimulated MAP kinase activity, suggesting that H2O2 induces MAP kinase activation via both PKC-dependent and PKC-independent pathways. Western analysis using a phosphotyrosine monoclonal antibody revealed increased tyrosine phosphorylation of proteins with approximate molecular weights of 72 and 125 kD after H2O2 exposure, demonstrating that H2O2 can stimulate the tyrosine phosphorylation of multiple cytosolic proteins, including MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrogen peroxide stimulates mitogen-activated protein kinase in bovine tracheal myocytes: implications for human airway disease. 794 86

Peroxisomes, isolated from homogenates of rat hearts (myocard), contain a beta-oxidation enzyme system which is indistinguishable from that found in liver, but the total capacity of beta-oxidation is only 0.8% of the liver value (expressed per g of tissue). Fatty acyl-CoA oxidase was assayed by an H2O2 based fluorescent assay avoiding important interfering side reactions. The presence of polypeptides with electrophoretic and immunological properties similar to the beta-oxidation enzymes of liver peroxisomes, was demonstrated by immunoblotting using polyclonal antibodies. The level of 72 and 52 kDa subunits of fatty acyl-CoA oxidase (FAO), quantitated by an anti-FAO1-16 peptide antibody, was only 1% of the level in liver (expressed per g of tissue). Immunoblots of one-dimensional (1-D) SDS-PAGE of rat heart and liver peroxisomal fractions revealed a 60 kDa subunit of the fatty acyl-CoA oxidase in addition to the known 72 and 52 kDa subunits. Immunoblots of two-dimensional (2-D) IEF/SDS-PAGE revealed that all subunits are strongly basic polypeptides, with a microheterogeneity, which probably represents deamidations of the polypeptides. The 2-D immunoblot also revealed another group of polypeptides with M(r) 72 kDa of less basic isoelectric point, possibly representing an isoform of fatty acyl-CoA oxidase. Substrate specificity studies revealed the highest Vmax values with C10-C12. For the very long-chain fatty acids C20-C24, the monoenes revealed much higher Vmax values than the saturated fatty acids. Administration of the classical peroxisome proliferator clofibrate resulted in a similar increase in the fatty acyl-CoA oxidase activity and the 72 and 52 kDa subunits of FAO in the heart. The response (activity) was found to change from 2.2-fold increase in young (34 days) to 11.1-fold increase in adult (76 days) rats. In contrast to liver, where the ratio of the increase in FAO mRNA to the increase in FAO activity was about 4, this ratio in heart was about 0.5.
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PMID:The peroxisomal beta-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate. 794 33

Albumin-gold complex (Alb-Au) was previously shown to bind selectively to plasmalemmal vesicles of capillary endothelium. Based on these findings, as well as on the ability of lactoperoxidase (LPO) to mediate the radioiodination of proteins, we have prepared a complex of gold particles bearing both albumin and anionized lactoperoxidase (Alb-Au-aLPO). The complex had a pI of 5.8, largely preserved aLPO enzymatic activity (approximately 74%), and was able to catalyze protein radioiodination. Upon washing out the blood, the complex was perfused in the mouse lung, the excess tracer removed, and a Na125I/H2O2 solution was introduced in the vasculature. After extensive washing, lung fragments were processed for either electron microscopy (EM), or to prepare a membrane-enriched fraction. In control experiments, lungs were perfused with native LPO (pI 9.3), or with a LPO-Affi Gel conjugate and further radioiodinated as described for Alb-Au-aLPO. By EM, it was found that both in tissue and in the isolated membrane fraction, only Alb-Au-aLPO labeled markedly and preferentially some uncoated pits and most plasmalemmal vesicles. Analysis by SDS-PAGE and autoradiography of a membrane-enriched fraction prepared from lungs perfused with Alb-Au-aLPO had some major identified 125I-labeled polypeptides of apparent molecular masses of 16, 18, 31, 36, 55, and 77 kDa. A different subset of polypeptides was labeled in lungs perfused with LPO, whereas after administration of LPO-Affi Gel the major radiolabeled polypeptides had a molecular mass of 33, 55 kDa and several peptides in the range of 77 to 160 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A method for selective radiolabeling of lung endothelium plasmalemmal vesicles, in situ. 795 13

L-Tryptophan 2',3'-oxidase is a novel enzyme that specifically catalyzes the alpha,beta-dehydrogenation of L-tryptophan derivatives. It was extracted from Chromobacterium violaceum and purified 108-fold to apparent homogeneity with a 34% overall recovery. The molecular weight of the native enzyme is approximately 680,000, and its isoelectric point is nearly equal to 4. SDS-polyacrylamide gel electrophoresis showed that the enzyme is composed of two components with molecular weight of approximately 74,000 and 14,000. It also contains a noncovalently bound heme prosthetic group, as judged from a typical spectrum showing maxima at 427, 530, and 560 nm. The catalyzed reaction is completed without side-product formation over a broad pH range comprised between 3 and 8. The enzyme is reoxidized at the expense of molecular oxygen by producing one molecule of H2O2. Kinetic parameters for modification of N-acetyl-L-tryptophanamide were determined (Km = 19.5 microM; kcat = 45.2 s-1). A Hill coefficient of about 1 suggests the absence of any cooperative effect. As inferred from both its kinetic and stability optimal conditions, L-tryptophan 2',3'-oxidase constitutes a promising tool for chemical modification of tryptophanyl side chain in peptides and proteins.
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PMID:Purification and partial characterization of an amino acid alpha,beta- dehydrogenase, L-tryptophan 2',3'-oxidase from Chromobacterium violaceum. 802 79

Estrogen induced peroxidase (EIP) activity revealed as a diaminobenzidine-H2O2 product in electron micrographs is apparent within cisternae of the RER and in large dilated apical vesicles, of which only a few have been seen to open into the uterine lumen. EIP activity is infrequently present in the trans cisternae of the rather diminutive Golgi complex in uterine epithelial cells from 12-72 h after treatment with estrogen. EIP activity is, however, prominent on the surface of microvilli of epithelial cells and as deposits in the lumen. Desalted uterine fluid (72 h) isolated by rotofor (Biorad) and analysed on isoelectric focusing gels that were stained with the diaminobenzidine-H2O2 reaction, reveal the existence of at least 6 peroxidase isoelectric variants with isoelectric points between pI 3.5 and pI 5.0. In similar preparations doubly-stained by diaminobenzidine-H2O2 and silver, peroxidase positive bands are enhanced, along with other isoelectric variants in the acidic pI range. In SDS-PAGE preparations, five prominent proteins ranging from 29-115 kD are present in 72 h uterine fluid. Luminal EIP coats the surface microvilli of the reproductive tract cells, and of viable spermatozoa incubated in uterine fluid. Peroxidase coated bacteria appear to be in the process of decay, and enzyme activity is present on the surface of most spermatozoa. It is not yet determined if EIP has bactericidal, spermicidal or capacitation functions.
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PMID:Estrogen induced peroxidase secretion from the endometrial epithelium: a possible function for the luminal enzyme. 808 2

Radiolysis of haemoglobin was carried out in phosphate buffer under air, N2 or N2O and with and without ethanol. Radiation products were separated by SDS-PAGE. The loss of subunits and simultaneous aggregation and fragmentation of haemoglobin was measured, if OH-radicals were unscavenged. There was no sensitizing effect of oxygen on the degradation process. Radiation-induced fragmentation was not a random process, but produced specific fragments. The estimated molecular weights of these fragments gave further support to the assumption that the aminoacyl-proline peptide group is the preferential breaking site if OH radicals react with proteins in the presence of oxygen. In contrast with lactate dehydrogenase and bovine serum albumin such fragmentation was observed not only after aerobic radiolysis but also under anaerobic conditions. This difference must be caused by the Feporphyrin system which reacts with H2O2 under release of oxygen. If haemoglobin was irradiated under air the yield of aggregates was much lower than under N2O or N2.
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PMID:Oxygen effect in the radiolysis of proteins. III. Haemoglobin. 810 37


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