| 1. |
- Christakopoulos, Paul, et al.
(author)
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Purification and characterization of a less randomly acting endo-1,4-beta-D-glucanase from the culture filtrates of Fusarium oxysporum
- 1995
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 316:1, s. 428-433
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Journal article (peer-reviewed)abstract
- An extracellular endo-1,4-β-D-glucanase from Fusarium oxysporum was purified by affinity chromatography and gel filtration. The enzyme purified in this way was homogeneous when judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. The protein corresponded to a molecular mass and pI value of 41.7 kDa and 6.4, respectively. It was optimally active at pH 4.5 and at 55°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and unsubstituted and substituted cello-oligosaccharides but was inactive on Avicel, filter paper, xylan, cellobiose, p-nitrophenyl-β-D-glucoside, and p-nitrophenyl-β-D-xyloside. However, the enzyme effected only a small change in viscosity of CMC per unit increase of reducing sugar. When cellotriose, cellotetraose, and cellopentaose were used as substrates, the enzyme released mainly cellobiose. Use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the glycosidic bond adjacent to 4-methylumbelliferone. Thus, the purified enzyme appeared to be a less randomly acting endoglucanase.
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| 2. |
- Christakopoulos, Paul, et al.
(author)
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Purification and mode of action of an alkali-resistant endo-1,4-β-glucanase from Bacillus pumilus
- 1999
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 364:1, s. 61-66
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Journal article (peer-reviewed)abstract
- Alkaline endo-1,4-β-d-glucanase was secreted byBacillus pumilusgrown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pIvalues of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0–8.0 and 60°C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-β-d-glucoside, 4-nitrophenyl-β-d-cellobioside, and 4-nitrophenyl-β-d-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.
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| 3. |
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| 4. |
- Landberg, Eva, et al.
(author)
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Glycosylation of Bile-Salt-Stimulated Lipase from Human Milk : Comparison of Native and Recombinant Forms
- 1997
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 344:1, s. 94-102
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Journal article (peer-reviewed)abstract
- Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19–26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types ofN-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short typeO-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.
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| 5. |
- Parmryd, I, et al.
(author)
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In vivo prenylation of rat proteins : modification of proteins with penta- and hexaprenyl groups.
- 1999
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 364:2, s. 153-60
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Journal article (peer-reviewed)abstract
- In vivo protein prenylation was studied in newborn rats by repeated injections of [3H]mevalonate. The highest level of protein-bound mevalonate metabolites was found in the kidney, but incorporation was observed in all organs studied. After fluorography of SDS-polyacrylamide gel electrophoresis-separated polypeptides, labeling was found in the 21- to 28-kDa molecular mass region and, after prolonged exposure of the film, additional bands at both higher and lower molecular masses could be detected. Protein prenylation in the kidney increased during the first 5 days after birth, whereas that in the liver reached a maximum on the fourth day. After methyliodide treatment of the prenylated proteins, farnesol, geranylgeraniol, and two larger isoprenoids, pentaprenol and hexaprenol, were released. In the liver, the ratio of protein-bound geranylgeraniol to farnesol increased from 2 to 4.5 during the first 5 days after birth. Upon subfractionation of the kidney, the highest level of labeling was found in mitochondria and microsomes. When the mitochondria were subfractionated into outer membranes, intermembrane space and an inner membrane/matrix fraction, the labeling pattern of prenylated polypeptides differed in all fractions. The results demonstrate that in vivo labeling of rats can be performed to study the extent, type, and distribution of protein prenylation.
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| 6. |
- Parmryd, I, et al.
(author)
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Protein prenylation in spinach--tissue specificity and greening-induced changes.
- 1997
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 339:1, s. 73-8
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Journal article (peer-reviewed)abstract
- Etiolated spinach seedlings, as well as petioles and blades of leaves of green seedlings, were labeled with [3H]mevalonate to study protein prenylation in several plant developmental stages. The polypeptide prenylation pattern of the leaf petiole and the leaf blade differed considerably, although some prenylated proteins were present in both tissues. During greening several prenylated polypeptides in the 30- to 46-kDa molecular mass region and two at 15 kDa became more abundant, while others in the 21.5- to 30-kDa region and one at 62 kDa showed a relative decrease. However, the relative amount of several of the prenylated polypeptides did not appear to be altered during the greening process. In etiolated seedlings, more thioether-linked farnesol than geranylgeraniol was found, whereas in seedlings grown under normal light conditions the converse situation prevailed.
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| 7. |
- Sigfridsson, Kalle, et al.
(author)
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Electron transfer in ruthenium-modified spinach plastocyanin mutants
- 1998
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In: Archives of Biochemistry and Biophysics. - : Academic Press. - 0003-9861 .- 1096-0384. ; 351:2, s. 197-206
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Journal article (peer-reviewed)abstract
- Four site-directed mutants of spinach plastocyanin, Pc(Leu12His), Pc(Leu15His), Pc(Thr79His), and Pc(Lys81His), have been modified by covalent attachment of a photoactive [Ru(bpy)2(im)]2+ complex at the surface-exposed histidine residues. The Pc-Ru complexes were characterized with optical absorption, CD, and EPR spectroscopy and their spectra were found to be similar to the unmodified proteins except in the case of the Pc(Leu12His) mutant which lost the Cu ion irreversibly during the Ru modification. Electron transfer (ET) within the other Pc-Ru complexes was studied with time-resolved optical spectroscopy, using an external-quencher approach. The fully reduced [Cu(I), Ru(II)] proteins were photoexcited and subsequently oxidized by an external quencher, [Ru(NH3)6]Cl3, forming the [Cu(I), Ru(III)] proteins. This was followed by an internal ET from Cu(I) to Ru(III). The rates of the internal ET reactions exhibit an exponential dependence on metal-to-metal separation, with a decay factor of 1.1 A-1. From a temperature-dependence study of the Ru-modified Pc(Lys81His) protein, a reorganization energy for the Cu-to-Ru ET reaction of 1.2 eV was determined. In this analysis it was found necessary to include an appreciable temperature dependence in the driving force of the ET reaction.
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| 9. |
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| 10. |
- Berna, Nathalie, et al.
(author)
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Cosolvent-induced adsorption and desorption of serum proteins on an amphiphilic mercaptomethylene pyridine-derivatized agarose gel
- 1996
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In: Arch Biochem Biophys. - : Elsevier BV. - 0003-9861. ; 330:1, s. 188-192
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Journal article (peer-reviewed)abstract
- We studied the effects of the following cosolvents on the adsorption and desorption of serum proteins from an amphiphilic mercaptomethylene pyridine-derivatized agarose gel: glucose, sucrose, polyethylene glycol (PEG), 2-methyl-2,4-pentanediol (MFD), sorbitol, pentaerythritol, glycerol, and Na2SO4. The water-structuring salt 0.4 M Na2SO4 was the most potent promoter of protein adsorption, followed by 5 M sorbitol and, to a lesser extent, 0.2 M PEG 1000 and 2.25 M MPD. The other cosolvents (4 M glucose, 1.5 M sucrose, 0.3 M pentaerythritol, and 7.6 M glycerol) were unable to promote protein adsorption to the gel. Attempts to modulate the salt-promotion effect of Na2SO4 with different cosolvents demonstrated the occurrence of synergistic effects for pentaerythritol, sorbitol, and glucose and antagonistic effects for the other cosolvents. Sorbitol and glycerol were found to be the most interesting co-solvents studied, as the first promoted protein adsorption, whereas the other disrupted protein interaction. As a consequence of these novel findings we propose sorbitol and glycerol, both well-known protein stabilizers, as possible alternatives to water-structuring salts during the adsorption phase and to deleterious organic solvents during the desorption phase on amphiphilic gels.
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| 11. |
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| 12. |
- Burnett, D, et al.
(author)
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Synthesis and secretion of procathepsin B and cystatin C by human bronchial epithelial cells in vitro: modulation of cathepsin B activity by neutrophil elastase
- 1995
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 317:1, s. 305-310
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Journal article (peer-reviewed)abstract
- Procathepsin B and cystatin C are found in human lung secretions. We investigated the capacity of human bronchial epithelial cells to synthesize and secrete these proteins. Immunoprecipitation of [35S]methionine-labeled proteins from cultured bronchial epithelial cell lysates, followed by denaturing gel electrophoresis and autoradiography, showed the presence of newly synthesized procathepsin B of M(r) 42,000; no mature form was detected. Cathepsin B in conditioned medium from epithelial cells was tagged with benzyloxycarbonyl-125I-tyrosyl-alanine-diazomethane before and after treatment of the medium with neutrophil elastase. Control medium again showed a predominant form of cathepsin B with a M(r) of 42,000, but upon treatment with neutrophil elastase this protein was converted to a M(r) of 38,000, similar to the active form previously found in lung secretions, and cathepsin B activity was generated. The medium also contained the cathepsin B inhibitor, cystatin C, but cystatins A, B, S, SN, SA, and kininogen were not detected. After removal of cystatin C from the medium, elastase was still required to activate procathepsin B. These results suggest that bronchial epithelial cells are a source of procathepsin B and cystatin C in lung secretions. Cleavage both of cystatin C and procathepsin B by neutrophil elastase is essential for the generation of cathepsin B activity in the medium.
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| 23. |
- Pavlov, MY, et al.
(author)
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Rate of translation of natural mRNAs in an optimized in vitro system
- 1996
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In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. - : ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS. - 0003-9861. ; 328:1, s. 9-16
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Journal article (other academic/artistic)abstract
- We report results on in vitro translation of an mRNA coding for elongation factor TuB which was in vitro transcribed from the tufB gene from Escherichia coil. Translation occurs at a rate of about 10 codons per second, which is close to the in vivo rate.
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