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11.
E. coli was found to grow anaerobically on lactate in the presence of trimethylamine N-oxide (TMANO), reducing it to trimethylamine. Anaerobic growth on glucose was promoted in the presence of TMANO. When a culture grown in complex medium was transferred to defined medium, growth on glucose and ammonia took place in the presence of TMANO after consumption of complex nutrients introduced with the preculture, in contrast to growth in nitrate respiration. The amounts of ethanol, succinate, and lactate among the fermentation products were decreased and that of acetate was increased in the presence of TMANO. Formate generation was much reduced at pH 7.4, whereas stoichiometric formation of formate was observed in the absence of TMANO. Cells grown anaerobically in the presence of TMANO had a higher activity of amine N-oxide reductase than cells grown under other conditions. The content of cytochrome-558 was elevated in the presence of TMANO during growth in complex medium. Cytochrome c-552 found in cells grown in diluted complex medium or defined medium in the presence of TMANO was oxidized by TMANO in cell extracts. The molar growth yield on glucose was higher in the presence of TMANO than in its absence and lower than that in the presence of nitrate. 相似文献
12.
Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding 总被引:4,自引:0,他引:4
Prostaglandin endoperoxide (PGH) synthase has a single iron protoporphyrin IX which is required for both the cyclooxygenase and peroxidase activities of the enzyme. At room temperature, the heme iron is coordinated at the axial position by an imidazole, and about 20% of the heme iron is coordinated at the distal position by an imidazole. We have used site-directed mutagenesis to investigate which histidine residues are involved in PGH synthase catalysis and heme binding. Individual mutant cDNAs for ovine PGH synthases were prepared with amino acid substitutions at each of 13 conserved histidines. cos-1 cells were transfected with each of these cDNAs, and the cyclooxygenase and peroxidase activities of the resulting microsomal PGH synthases were measured. Mutant PGH synthases in which His-207, His-309, or His-388 was replaced with either glutamine or alanine lacked both activities. Gln-386 and Ala-386 PGH synthase mutants exhibited cyclooxygenase but not peroxidase activities. Other mutants exhibited both activities at varying levels. Because binding of heme renders native PGh synthase resistant to cleavage by trypsin, we examined the effects of heme on the relative sensitivities of native, Ala-204, Ala-207, Ala-309, Ala-386, and Ala-388 mutant PGH synthases to trypsin as a measure of the heme-protein interaction. The Ala-309 PGh synthase mutant was notably hypersensitive to tryptic cleavage, even in the presence of exogenous heme; in contrast, the native enzyme and the other alanine mutants exhibited similar, lower sensitivities toward trypsin and, except for the Ala-386 mutant, were partially protected from trypsin cleavage by heme. Preincubation of the native and each of the alanine mutant PGH synthases, including the Ala-309 mutant, with indomethacin protected the proteins from trypsin cleavage. Thus, all the mutant proteins retain sufficient three-dimensional structure to bind cyclooxygenase inhibitors. Our results suggest that His-309 is one of the heme ligands, probably the axial ligand, of PGH synthase. Two other histidines, His-207 and His-388, are essential for both PGH synthase activities suggesting that either His-207 or His-388 can serve as the distal heme ligand; however, the trypsin cleavage measurements imply that neither His-207 nor His-388 is required for heme binding. This is consistent with the fact that only 20% of the distal coordination position of the heme iron of PGH synthase is occupied by an imidazole side chain. 相似文献
13.
Aspirin selectively acetylates Ser-530 of prostaglandin endoperoxide (PGH) synthase-1. This causes inactivation of the cyclooxygenase activity of the enzyme, but does not appreciably affect its peroxidase activity. Although the aspirin-acetylated enzyme is inactive, we found that PGH synthase-1 in which Ser-530 had been replaced with an alanine was catalytically active; accordingly, we proposed that aspirin inhibits cyclooxygenase activity by placing a larger than normal side chain at position 530 thereby interfering with arachidonate binding (DeWitt, D.L., El-Harith, E. A., Kraemer, S. A., Andrews, M. J., Yao, E. F., Armstrong, R. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 5192-5198). As a further test of this hypothesis we have used site-directed mutagenesis and transient expression in cos-1 cells to prepare and characterize five additional substitutions of Ser-530. Consistent with our proposal, the presence of amino acids with bulky side chains at position 530 inhibited cyclooxygenase activity and decreased the apparent affinity of the enzyme for arachidonate. In related work, we characterized a series of mutant PGH synthases-1 having substitutions at residues adjoining Ser-530, including Phe-529, Leu-531, Lys-532, and Gly-533, in order to evaluate the contributions of each residue to cyclooxygenase catalysis. The most significant conclusion of this part of the study is that residues 529-533 all are important for the peroxidase activity as well as the cyclooxygenase activity of PGH synthase-1. Phe-529, in particular, was found to be critical for PGH synthase-1 structure and catalysis; some substitutions at this position led to the production of proteins lacking about 100 amino acids from their COOH termini. 相似文献
14.
Sachio Hayashi Masaharu Nonoguchi Yoshihiko Shimokawa Mikihiko Tubouchi Yoshiyuki Takasaki Kiyohisa Imada 《Journal of industrial microbiology & biotechnology》1992,9(2):145-147
Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK
m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK
m values ofE-2 andP-2. 相似文献
15.
Effects of Phosphate in Medium on Protein Secretion in a Protein-Producing Bacterium, Bacillus brevis 47 总被引:2,自引:0,他引:2 下载免费PDF全文
Norihiro Tsukagoshi Hidehiko Yamada Akio Tsuboi Shigezo Udaka 《Applied microbiology》1981,42(2):370-374
Bacillus brevis 47 secreted up to 1 mg of protein per ml in a chemically defined medium, depending on phosphate concentration. The composition of exoproteins was altered quantitatively by the concentration of external phosphate. Morphologically, B. brevis 47 showed a distinct three-layered cell wall structure and shed the outer two layers during growth. 相似文献
16.
Tokimatsu Koji Tang Longlong Yasuoka Rieko Ii Ryota Itsubo Norihiro Nishio Masahiro 《The International Journal of Life Cycle Assessment》2020,25(9):1710-1736
The International Journal of Life Cycle Assessment - Despite the long-standing demand for research on dynamic lifecycle assessment (LCA) for policymaking, only a few studies have addressed this... 相似文献
17.
18.
Noriko Umemoto Yuhei Nishimura Yasuhito Shimada Yukiko Yamanaka Seiya Kishi Saki Ito Kana Okamori Yuuki Nakamura Junya Kuroyanagi Zi Zhang Liqing Zang Zhipeng Wang Norihiro Nishimura Toshio Tanaka 《Molecular biotechnology》2013,55(2):131-142
A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases. 相似文献
19.