Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
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Laser photolysis techniques have been used to characterize the reactivity of triplet state lipoidal benzophenone derivatives toward fatty acids and glycerides in benzene solution. The reactivities of benzophenone-4-heptyl-4'-pentanoic acid (BHPA) toward fatty acid compounds having different configurations of olefinic bonds have been determined. The rates of hydrogen abstraction are found to be lower when compared with similar measurements using benzophenone alone. However, the contribution of physical quenching of the triplet derivative by double bonds also appears to be slightly lower than that found with benzophenone itself. The hydrogen abstraction efficiencies of three other benzophenone derivatives toward linoleic acid in benzene have also been measured. For benzophenone incorporated into a fatty acid molecule, there is a limited relationship between structure and photoreactivity. Finally, these sensitizers have been incorporated into mixed SDS/linoleate micelles to determine the effects of molecular organization on photochemical behavior of the sensitizer and lipid.
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PMID:Hydrogen abstraction from lipids by triplet states of derivatized benzophenone photosensitizers. 234 58

The heat shock response was studied as a model for control of gene expression and protein synthesis in Giardia lamblia. Cultured trophozoites were metabolically labelled with [35S]methionine, and proteins were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. A temperature shift from 37 degrees C to 43 degrees C resulted in the depression of normal protein synthesis, and the enhanced synthesis of four major heat shock proteins of 100, 83, 70 and 30 kDa. This response resembles that seen in other organisms of wide phylogenetic diversity. An examination of the kinetics of induction and recovery from heat shock suggests that the individual heat shock proteins are independently regulated. In vitro translation of messenger RNA isolated from heat shocked cells further indicates that regulation occurs at both transcriptional and translational levels. The response of G. lamblia to other stresses including cysteine deprivation, exposure to oxygen, ethanol, hydrogen peroxide, and the chemotherapeutic drugs metronidazole and quinacrine was also investigated. The induction of two or more of the heat shock proteins was generally observed; however, certain treatments inhibited synthesis of all proteins including heat shock proteins.
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PMID:Heat shock and stress response in Giardia lamblia. 245 80

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.
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PMID:Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated. 255 70

The high molecular weight 12 S protein from rape seed was isolated in a homogeneous form and characterized. Six subunits were isolated by PAGE in the presence of SDS and 0.2 M 2-mercaptoethanol. These subunits (s1 to s6) were found in the protein in the weight ratio of 1.32:1.2:1.15:1.0:1.21:1.11. The molecular weights and first two N-terminal amino acids of the isolated subunits were 64,800 and phenylalanine, alanine (s1), 50,650 and valine, tyrosine (s2), 42,500 and phenylalanine, leucine (s3), 28,800 and threonine, glutamic acid (s4), 19,100 and cystine, isoleucine (s5) and 15,600 and alanine, phenylalanine (s6). The number of side chain carboxyl, imidazole and epsilon-amino groups were calculated from the hydrogen ion titrations, which were in agreement with the amino acid assay. Besides, the N-terminal amino acid sequence upto 43 residues for one subunit (s6) is reported using Edman degradation.
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PMID:Hydrogen ion titration of 12 S rape seed protein and partial N-terminal sequence of one of it's subunits. 261 53

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity. 1. Two-chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]noradrenaline release and binding to rat brain membranes. 2. Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N-terminal moiety of the heavy chain, called fragment beta 2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C-terminal part of the heavy chain. 3. The heavy chain alone inhibited K+-evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration-response ratio was flat and 10-100-fold higher concentrations were required than with native or reconstituted two-chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two-chain toxin increased it. Binding to rat brain membranes was assessed by competition with 125I-labelled two-chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two-chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000-fold lower affinity than native or reconstituted two-chain toxin. The light chain did not bind to membranes in either test. Our data indicate that (a) the light chain and the N-terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two-chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.
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PMID:Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties. 275 37

Functional and biochemical techniques were used to further characterize heterogeneity between rat Kupffer cells and peritoneal macrophages. Both macrophage cell types were found to phagocytize antibody coated sheep red blood cells in a time-dependent manner. However, Kupffer cells were two to three times more phagocytic than were peritoneal macrophages. In contrast, the peritoneal cells released significantly more superoxide anion in response to the complement cleavage product, C5a and the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate, and produced more hydrogen peroxide than did the liver macrophages. Both cell types responded chemotactically to C5a. These results suggest that macrophages may develop specialized functions depending on the needs of their local environment. Using one and two dimensional SDS-polyacrylamide gel electrophoresis, we also compared the production of newly synthesized proteins by Kupffer cells and peritoneal macrophages. In general, the macrophages were found to produce similar types and numbers of proteins with some exceptions. These included proteins that were unique to peritoneal macrophages and other proteins observed only in Kupffer cells. The production of these proteins in liver macrophages did not appear to correlate with levels of functional activation, but may be more related to the tissue origin of the cells.
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PMID:Functional and biochemical properties of rat Kupffer cells and peritoneal macrophages. 284 98

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.
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PMID:Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola. 284 44

When cytochrome c oxidase is incubated at 43 degrees C for approximately 75 min in a solution containing the zwitterionic detergent sulfobetaine 12, the CuA site is converted into a type II copper as judged by changes in the 830-nm absorption band and the EPR spectrum of the enzyme. SDS-PAGE and sucrose gradient ultracentrifugation indicate concomitant loss of subunit III and monomerization of the enzyme during the heat treatment. Comparison of the optical and resonance Raman spectra of the heat-treated and native protein shows that the heme chromophores are not significantly perturbed; the resonance Raman data indicate that the small heme perturbations observed are limited to the cytochrome a3 site. Proton pumping measurements, conducted on the modified enzyme reconstituted into phospholipid vesicles, indicate that these vesicles are unusually permeable toward protons during turnover, as previously reported for the p-(hydroxymercuri)benzoate-modified oxidase and the modified enzyme obtained by heat treatment in lauryl maltoside. The sulfobetaine 12 modified enzyme is no longer capable of undergoing the recently reported conformational transition in which the tryptophan fluorescence changes upon reduction of the low-potential metal centers. Control studies on the monomeric and subunit III dissociated enzymes suggest that the disruption of this conformational change in the heat-treated oxidase is most likely associated with perturbation of the CuA site. These results lend support to the suggestion that the fluorescence-monitored conformational change of the native enzyme is initiated by reduction of the CuA site [Copeland et al. (1987) Biochemistry 26, 7311].
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PMID:Conversion of CuA to a type II copper in cytochrome c oxidase. 285 58

The absorbance and fluorescence spectral properties of mitochondrial F1-ATPase confirm that this protein does not contain tryptophan residues and therefore its fluorescence is due to tyrosines. The 36% increase in the fluorescence and the almost 100% increase in quantum yield upon denaturation of the protein suggest that a considerable number of tyrosyl residues have a very low quantum yield in the native enzyme. Quenching experiments using iodide indicate that all of the fluorophores are quenched and also all of them with the same quenching constant. These observations are interpreted as confirmatory of what has been found with several other proteins whose fluorescence originates from tyrosyl residues, where the buried tyrosines fluoresce with a much lower quantum yield than those which are exposed. ATP added to F1 previously depleted of loosely bound nucleotides changes the quenching constant of iodide and the quantum yield and this is interpreted to be due to a conformational change induced by the binding of the nucleotide to the enzyme. Addition of 2-mercaptoethanol decreases, although slightly, the polarization of the fluorescence. However, SDS addition gives a much bigger decrease. Hence disulphide bridges are less important for the tertiary structure of the protein than hydrophobic interactions, hydrogen bonding or other forces. Nevertheless the conformational change induced by reduction of disulphide bridges is detected in iodide quenching experiments and the change of the quantum yield of the enzyme.
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PMID:Structural studies of mitochondrial coupling factor 1 using tyrosine fluorescence. 286 Nov 23

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.
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PMID:Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16. 308 7


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