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141.
142.
Hirai K Katayama Y Peltre G Tsuji S 《Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society》2008,46(1):111-116
In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR) was attempted, using d-tubocurarine (d-TC), a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG) for acetylcholine (ACh) and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA), a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC) to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB) precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest) of the postsynaptic membrane (between the junctional folds) which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm), while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex. 相似文献
143.
144.
Yoshifumi Maeda Atsuhide Yagyu Akihiko Sakurai Yutaka Fujii Hiroyuki Uchida 《World journal of microbiology & biotechnology》2008,24(6):797-804
Bacterium MEY43, an isolate from soil, produced aldehyde oxidase when it was cultivated in a medium containing methanol as
a sole source of carbon and energy. The methylotrophic bacterium was identified as Brevibacillus sp. Its cultivation in media containing other substrates, such as ethanol and glucose, resulted in little production of the
enzyme. Aldehyde oxidase purified from a cell-free extract of the bacterium was a hetero-trimeric protein comprised of large,
medium, and small subunits with molecular masses of 87, 35, and 19 kDa, respectively. Its UV/visible spectrum and the presence
of molybdenum, 5′-CMP, flavin adenine dinucleotide, iron, and acid-labile sulfur suggested that the enzyme belonged to the
xanthine oxidase family. The enzyme acted on a wide range of aliphatic and aromatic aldehydes. The K
m value for formaldehyde was 32 mM, whereas those for the other aldehydes tested were below 0.2 mM. When 10 mM glutaraldehyde
was treated with 2.0 units of the enzyme ml−1 in the presence of 100 units ml−1 catalase for 120 min, the concentration of the aldehyde decreased to below a detectable level. 相似文献
145.
Susumu Ichiyama Tatsuo Kurihara Yoshifumi Kogure Susumu Tsunasawa Haruhiko Kawasaki Nobuyoshi Esaki 《Biochimica et Biophysica Acta - Proteins and Proteomics》2004,1698(1):27-36
Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation. 相似文献
146.
Aerobic and Anaerobic Toluene Degradation by a Newly Isolated Denitrifying Bacterium, Thauera sp. Strain DNT-1 总被引:4,自引:0,他引:4
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147.
148.
Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47. 总被引:5,自引:0,他引:5
Takaki Koide Yoshifumi Takahara Shinichi Asada Kazuhiro Nagata 《The Journal of biological chemistry》2002,277(8):6178-6182
HSP47 is an essential procollagen-specific molecular chaperone that resides in the endoplasmic reticulum of procollagen-producing cells. Recent advances have revealed that HSP47 recognizes the (Pro-Pro-Gly)(n) sequence but not (Pro-Hyp-Gly)(n) and that HSP47 recognizes the triple-helical conformation. In this study, to better understand the substrate recognition by HSP47, we synthesized various collagen model peptides and examined their interaction with HSP47 in vitro. We found that the Pro-Arg-Gly triplet forms an HSP47-binding site. The HSP47 binding was observed only when Arg residues were incorporated in the Yaa positions of the Xaa-Yaa-Gly triplets. Amino acids in the Xaa position did not largely affect the interaction. The recognition of the Arg residue by HSP47 was specific to its side-chain structure because replacement of the Arg residue by other basic amino acids decreased the affinity to HSP47. The significance of Arg residues in HSP47 binding was further confirmed by using residue-specific chemical modification of types I and III collagen. Our results demonstrate that Xaa-Arg-Gly sequences in the triple-helical procollagen molecule are dominant binding sites for HSP47 and enable us to predict HSP47-binding sites in homotrimeric procollagen molecules. 相似文献
149.
Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-γ-Glutamate
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Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a γ-linkage, called poly-γ-glutamate (γ-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for γ-PGA (designated γ-PGA hydrolase, PghP) from bacteriophage ΦNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed γ-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-γ-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn2+ or Mn2+ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated Mr of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel γ-glutamyl hydrolase. Whereas phage ΦNIT1 proliferated in B. subtilis cells encapsulated with γ-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce γ-PGA. Analogous to polysaccharide capsules, γ-PGA appears to serve as a physical barrier to phage absorption. Phages break down the γ-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells. 相似文献
150.