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1. 脂質およびプリン代謝障害による代謝性ミオパチー metabolic myopathies caused by disorders of lipid and purine metabolism
2. Structure and function of normal hemoglobins
3. サラセミアの臨床症状および診断 clinical manifestations and diagnosis of the thalassemias

English Journal

Viral potassium channels as a robust model system for studies of membrane-protein interaction.

Braun CJ1, Lachnit C1, Becker P1, Henkes LM2, Arrigoni C3, Kast SM2, Moroni A4, Thiel G1, Schroeder I5.Author information 1Membrane Biophysics, Technical University of Darmstadt, Schnittspahnstrasse 3, 64287 Darmstadt, Germany.2Physikalische Chemie III, Technische Universität Dortmund, Otto-Hahn-Str. 6, 44227 Dortmund, Germany.3Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milano, Italy.4Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milano, Italy; CNR-IBF, Via Celoria 26, 20133 Milano, Italy.5Membrane Biophysics, Technical University of Darmstadt, Schnittspahnstrasse 3, 64287 Darmstadt, Germany. Electronic address: schroeder@bio.tu-darmstadt.de.AbstractThe viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking.
Biochimica et biophysica acta.Biochim Biophys Acta.2014 Apr;1838(4):1096-103. doi: 10.1016/j.bbamem.2013.06.010. Epub 2013 Jun 17.
The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its
PMID 23791706

Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T.

Martínez-Gómez AI1, Soriano-Maldonado P1, Andújar-Sánchez M1, Clemente-Jiménez JM1, Rodríguez-Vico F1, Neira JL2, Las Heras-Vázquez FJ1, Martínez-Rodríguez S3.Author information 1Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain.2Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Complex Systems Physics Institute, 50009 Zaragoza, Spain.3Dpto. Química y Física, Universidad de Almería, Campus de Excelencia Internacional Agroalimentario, ceiA3, 04120 Almería, Spain; Centro de Investigación en Biotecnología Agroalimentaria, BITAL, Almería, Spain; Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain; Dpto. Química Física, Universidad de Granada, 18071 Granada, Spain. Electronic address: srodrig@ual.es.AbstractAllantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 °C. Optimal parameters for the enzyme activity were pH 7.5 and 50 °C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s(-1), respectively. Co(2+) proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase.
Biochimie.Biochimie.2014 Apr;99:178-88. doi: 10.1016/j.biochi.2013.12.002. Epub 2013 Dec 12.
Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial
PMID 24333989

New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

Santos A1, Mendes S, Brissos V, Martins LO.Author information 1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av da República, 2780-157, Oeiras, Portugal.AbstractThis work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.
Applied microbiology and biotechnology.Appl Microbiol Biotechnol.2014 Mar;98(5):2053-65. doi: 10.1007/s00253-013-5041-4. Epub 2013 Jul 3.
This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including
PMID 23820555