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41.
Molecular Characterization and Expression of Pyruvate Formate-Lyase-Activating Enzyme in a Ruminal Bacterium, Streptococcus bovis
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To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe2+ for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low. 相似文献
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Jun Moriya Masayoshi Sakakura Yuji Tokunaga R. Scott Prosser Ichio Shimada 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
The determination of protein–protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein–protein interface by NMR spectroscopy, 13C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct 13C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while 13C nuclei within the interior of the proteins may be distinguished by a lack of such shifts.Method
We measured the 13C NMR paramagnetic shifts induced by TEMPOL by recording 13C–13C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).Results
Upon complexation of ubiquitin with YUH1, 13C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data.Conclusions
The TEMPOL-induced 13C chemical shift perturbation is useful to determine precise protein–protein interfaces.General significance
The present method is a useful method to determine protein–protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities. 相似文献46.
To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD+-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation
factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate
(GAP), were not very different between GAPDH- and NADP+-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts
of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic
flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1,
JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD+ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly
controlled by LDH activity. 相似文献
47.
Toshihiko Sugiki Chie Yoshiura Yutaka Kofuku Takumi Ueda Ichio Shimada Hideo Takahashi 《Protein science : a publication of the Protein Society》2009,18(5):1115-1120
Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments. 相似文献
48.
Shunsuke Igarashi Masanori Osawa Shin-ichiro Ozawa Ichio Shimada 《Biomolecular NMR assignments》2010,4(1):17-20
HisJ is a histidine binding subunit of the histidine permease, which exists in the outer membrane of Gram-negative bacteria. In order to incorporate the periplasmic histidine into the cell, HisJ captures histidine in the periplasm, and transfers the histidine to the transmembrane complex of histidine permease that is an ABC transporter. We established the backbone resonance assignments of 1H/13C/15N-labeled HisJ from Escherichia coli, in the histidine-bound and unbound states. 相似文献
49.
Saito S Aoki I Sawada K Sun XZ Chuang KH Kershaw J Kanno I Suhara T 《Radiation research》2011,175(1):1-9
Our purpose was to noninvasively assess formation of the microvasculature, blood-brain barrier (BBB) and blood-CSF barrier formation of prenatal X-ray-induced CNS abnormalities using quantitative MRI. Eight pregnant female Sprague-Dawley rats were divided into two groups consisting of control and X-irradiated animals. After birth, 20 neonatal male rats were divided into four groups of five rats. To evaluate the development of the BBB, changes in T(1) induced by Gd-DTPA were compared quantitatively in normal and prenatally irradiated animals in the formative period 1 to 2 weeks after birth. To assess the abnormalities of the microvasculature, quantitative perfusion MRI and MR angiography were also used. Histology was also performed to evaluate the BBB (albumin) and vascular endothelial cells (laminin). Decreased cerebral blood flow (CBF) and angioarchitectonic abnormalities were observed in the prenatally irradiated rats. However, abnormalities of the BBB and blood-CSF barrier were not observed using Gd-enhanced MRI and albumin staining. Quantitative perfusion MRI, MR angiography and Gd-enhanced T(1) mapping are useful for assessing CNS disturbance after prenatal exposure to radiation. These techniques provide important diagnostic information for assessing the condition of patients during the early stages of life after accidental or unavoidable prenatal exposure to radiation. 相似文献
50.
Atsuyuki Tomizawa Itsuko Ishii Zhivko Zhelev Ichio Aoki Sayaka Shibata Mitsukazu Kitada Rumiana Bakalova 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011