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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca2+ uptake; from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min (mean +/- SE) (P less than 0.01) and Ca(2+)-ATPase activity from 2.08 +/- 0.05 mumol Pi/min.mg to 0.28 +/- 0.04 mumol Pi/min.mg (mean +/- SE) (P less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca(2+)-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H2O2 + Fe(2+)-EDTA) as well as H2O2 (12 mM) were without any effect on the 97,000 dalton Ca(2+)-ATPase band of sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Singlet oxygen: a potential culprit in myocardial injury? 131 3

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.
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PMID:Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms. 131 98

1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.
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PMID:Immunopurification of gastric parietal cell tubulovesicles. 131 5

A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.
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PMID:Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity. 132 Oct 48

A plasma membrane Ca(2+)-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca(2+)-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca(2+)-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca(2+)-pump ATPase. These results suggest that the direct phosphorylation of the Ca(2+)-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca(2+)-pump ATPase by G-kinase.
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PMID:Plasma membrane Ca(2+)-pump ATPase is not a substrate for cGMP-dependent protein kinase. 132 92

1. Purified (Na+ + K+)-ATPase was irreversibly inhibited upon exposure to hydrogen peroxide, the superoxide anion, and the hydroxyl radical. 2. Comparison of the SDS-gel electrophoretic patterns of the ATPase samples exposed to these oxidants revealed that inhibition occurred either without gross structural changes, or concomitant with fragmentation and cross-linking of the enzyme subunits. 3. The oxidant modified ATPase was also shown to be more susceptible to degradation by several proteolytic enzymes.
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PMID:(Na+ + K+)-ATPase: inactivation and degradation induced by oxygen radicals. 132 81

Stimulation of gastric acid secretion is mediated by cAMP which regulates the proton pump through an A-kinase-dependent phosphoprotein. The purpose of this study was to isolate a stimulation-dependent gastric phosphoprotein capable of stimulating acid secretion. Gastric glands were prepared from rabbit gastric mucosa and acid secretion was stimulated with cAMP. A detergent extract of these stimulated gastric membranes was fractionated by gel chromatography and assayed for functional activity by measurement of [14C]-aminopyrine accumulation in permeabilized resting gastric glands or measurement of H(+)-K(+)-ATPase activity in inhibited gastric microsomes. We hereby report isolation of a membrane-bound, A-kinase-dependent phosphoprotein which enhances aminopyrine accumulation in digitonin-permeabilized gastric glands (32%) and stimulates H(+)-K(+)-ATPase activity in gastric microsomes to a level 55% of the maximal stimulation observed in the presence of valinomycin. Incubation of this phosphoprotein with [32P]ATP and the catalytic subunit of A-kinase resulted in [32P] incorporation into a protein which coincided with a single protein band on SDS-PAGE (17,500 Da).
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PMID:Isolation of a gastric phosphoprotein which stimulates acid secretion. 132 65

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.
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PMID:Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes. 133 Oct 35

Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).
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PMID:DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes. 133 86

Na,K-ATPase was purified from pig and mink kidney. Enzyme batches of high specific activity apparently contained only alpha- and beta-peptides, judging from the SDS-gel-electrophoretic pattern. Careful determinations of the ouabain-binding capacity and binding kinetics were carried out. The question whether the functional unit is an alpha beta-protomer, a diprotomer or even a polymer was analysed from the binding stoichiometry and the binding pattern at nonsaturating ouabain concentrations. The binding capacity exceeded half of the theoretical maximum value for exclusively alpha beta-promoters. A half-of-the-sites reactivity of a diprotomer thus seems less likely, though nearly half of the protein is not accounted for in its absence. When ouabain binding took place in the presence of Na+, at least two binding components were noticed. Most probably the two components are not of equal size, thus excluding a co-operative diprotomer construction of the active ouabain-binding units. If the co-operativity model has to be abandoned, the phenomenon could be due to two or more isozymes exhibiting different Na(+)-affinities. Isoforms of the hydrolytic alpha-peptide were analysed by an ELISA technique and on blots of Na,K-ATPase peptides utilizing commercial isoform-specific antibodies. Though not absolutely specific, the individual antibodies were assumed to be monospecific with respect to the single isoforms of the alpha-peptide. In that case, data were consistent with a significant contribution of alpha 2- and alpha 3-isoforms (about 15%), in addition to the predominant alpha 1-isoform in kidney Na,K-ATPase.
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PMID:Heterogeneity of Na,K-ATPase from kidney. 133 54


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