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201.
Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method 总被引:1,自引:0,他引:1
A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids. By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified. This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source. The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system. Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand. The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization. 相似文献
202.
203.
Regulation of phosphorus stoichiometry and growth rate of consumers: theoretical and experimental analyses with Daphnia 总被引:1,自引:0,他引:1
Initial theories of ecological stoichiometry were based on the assumption that the mass-specific content of key nutrient elements
(such as P), changes little within a consumer species. However, evidence has shown that this content changes substantially
according to feeding conditions. To clarify how the specific P content (S
P) of a consumer species depends on food conditions and relates to the growth rate, we constructed a multiple mass-balance
model incorporating feeding and metabolic costs and stoichiometrically regulated releases for C and P. The validity of the
model was then tested experimentally by examining the growth rates and S
P of Daphnia pulicaria under various food conditions. The experimental observation agreed qualitatively well with the model, showing that the S
P of consumers relates positively to growth rate at high food C:P ratios but negatively at low food C:P ratios. Thus, within
a consumer species, individuals with high S
P do not necessarily grow at high rates. The concordance in results between the model and our observation suggests that maintenance
costs for both P and C are substantial regardless of food conditions and play crucial roles in determining the relationship
between the S
P and growth rate of consumers. 相似文献
204.
Shimizu T Ihara K Maesaki R Kuroda S Kaibuchi K Hakoshima T 《The Journal of biological chemistry》2000,275(24):18311-18317
Mg(2+) ions are essential for guanosine triphosphatase (GTPase) activity and play key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. We determined the crystal structure of a small GTPase RHOA complexed with GDP in the absence of Mg(2+) at 2.0-A resolution. Elimination of a Mg(2+) ion induces significant conformational changes in the switch I region that opens up the nucleotide-binding site. Similar structural changes have been observed in the switch regions of Ha-Ras bound to its guanine nucleotide exchange factor, Sos. This RHOA-GDP structure reveals an important regulatory role for Mg(2+) and suggests that guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation in GDP/GTP exchange. 相似文献
205.
Yoshitaka Shimizu Shinjiro Todo Shinsaku Imashuku 《Prostaglandins & other lipid mediators》1986,32(4)
To study the precise mechanism of cytotoxic activity of PGD2 or Δ12-PGJ2 (a biological active metabolite of PGD2), we examined the effect of various compounds on PGD2 or Δ12-PGJ2 cytottoxic, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD2 cytotoxicity on NCG cells. When Δ12-PGJ2 was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and α-amanitin did not affect PGD2 or Δ12-PGJ2 cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD2 or Δ12-PGJ2 exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD2 or Δ12-PGJ2. 相似文献
206.
Toru Shimizu Masahiro Hatano Seiji Nagao Yoshinori Nozawa 《Biochemical and biophysical research communications》1982,106(4):1112-1118
43Ca NMR spectra of Ca2+-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca2+ from Tet. CaM. is estimated to be approx. 2.7 × 103 s?1 under a certain assumption. Relaxation rates of 43Ca NMR of Ca2+-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca2+ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of 43Ca NMR signals are increased by adding excessive Mg2+ to the Ca2+-Tet. CaM. solutions. The addition of Mg2+ to the Ca2+-Tet. CaM. complex decreases apparent pKa value of the complex as well. 相似文献
207.
Saiga A Uozumi N Ono T Seno K Ishimoto Y Arita H Shimizu T Hanasaki K 《Prostaglandins & other lipid mediators》2005,75(1-4):79-89
Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated. 相似文献
208.
Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity. 相似文献
209.
A Hayashi T Suzuki A Shimizu K Imai H Morimoto 《Archives of biochemistry and biophysics》1968,125(3):895-901
210.
Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli 总被引:1,自引:0,他引:1
Shimizu T Shibata H Araya T Nakatsu T Miyairi K Okuno T Kato H 《Protein expression and purification》2005,44(2):558-135
Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation. 相似文献