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Midori Umekawa Kaito Hamada Naoto Isono Shuichi Karita 《Journal of Applied Glycoscience》2020,67(4):103
Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg 2+ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment. 相似文献
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Hexokinase PII (Hxk2) is a yeast glucose phosphorylating enzyme that, besides its role in glycolysis, seems to have an additional role in glucose signalling. To study the domains in Hxk2 that may participate in this latter process, we have constructed 11 mutant alleles using site-directed mutagenesis. Six of them were clustered charged-to-alanine mutants in which clusters of charged residues were changed to alanine residues. Two of them contained substitutions in Ser15 to either alanine or glutamic acid and three of them had deletions at either the N-terminus or the C-terminus of the protein. In most of them, the catalytic activity correlated directly with their functionality in glucose signalling. However, we found two mutants (Delta1-15 and Delta476-486) that, having low catalytic activity, were still fully functional in glucose signalling. This may indicate that other factors and not just the catalytic activity of the enzyme may be important for the functionality of the protein in glucose signalling. 相似文献
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The enzymatic steps involved in the inhibition of glycolysis by 2-deoxygalactose in Saccharomyces cerevisiae have been investigated. Yeast, incubated with 2-deoxygalactose, accumulates up to 8 mM-2-deoxygalactose, 30 mM-2-deoxygalactose-1-phosphate and 0.25 mM-UDP-2-deoxygalactose and UDP-2-deoxyglucose. An inverse correlation between 2-deoxygalactose-1-phosphate content and rate of glycolysis has been observed. The intracellular concentration of glycolytic intermediates and related metabolites point to the hexokinase and phosphofructokinase steps as the targets for the inhibition of glycolysis by 2-deoxygalactose and rule out all other mechanisms that have been proposed to explain this inhibition. 相似文献
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Human pancreatic glucokinase (GlkB, hexokinase IV) has been expressed in Saccharomyces cerevisiae. The recombinant protein showed similar enzyme kinetics to those described for the original enzyme. When expressed in hxk2 yeast mutants, GlkB complemented both the glucose induction and the glucose repression defects present in the mutant. It was also functional in regulating the activity of the Snf1 kinase complex in response to glucose, participating in the regulation of the Reg1/Glc7 phosphatase complex, as its yeast counterpart. 相似文献
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The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP. 相似文献
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The main energetic pathways, fermentation and respiration, and the general ion transport properties of Candida albicans were studied. Compared to Saccharomyces cerevisiae, we found that in C. albicans: (a) the cell mass yield when grown in YPD was significantly larger; (b) it required longer times to be starved of endogenous substrates; (c) ethanol production was lower but significant; (d) respiration was also lower; (e) it showed a small activity of an alternative oxidase; (f) fermentation and oxidative phosphorylation seemed to compete for both ADP and NADH; and (g) NADH levels were lower. Regarding ion transport and compared to S. cerevisiae: (a) the general mechanism was similar, with a plasma membrane H+‐ATPase that generates both a plasma membrane ΔpH and a ΔΨ, the latter being responsible for driving K+ inside; (b) its acidification capacity is slightly smaller and less sensitive to activation by high pH; and (c) the presence of K+ results in a large activation of both respiration and fermentation, most probably due to the energy required in the process. ADP produced by H+‐ATPase stimulation by high pH or the addition of K+ at low pH results in the increase of both respiration and fermentation. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
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Mizuno T Kishimoto T Shinzato T Haw R Chambers A Wood J Sinclair D Uemura H 《Yeast (Chichester, England)》2004,21(10):851-866
In the yeast two-hybrid system, the N-terminal region of Rap1p was shown to interact with Gcr1p and Gcr2p. Disruption of gcr1 and/or gcr2 in the two-hybrid reporter strain demonstrated that the interaction with Gcr1p does not require Gcr2p, whereas the interaction with Gcr2p is mediated through Gcr1p. Deletion of the N-terminal region of Rap1p alone did not show a growth phenotype, but a growth defect was observed when this mutation was combined with a gcr2 deletion. The poor growth of the gcr1 null mutant was not affected further by the N-terminal deletion of Rap1p, but the growth of gcr1 strains with mutations in the DNA binding region of Gcr1p was affected by the removal of the N-terminal region of Rap1p. These results suggest that one function of the N-terminal region of Rap1p, presumably the BRCT domain, is to facilitate the binding of Gcr1p to the promoter by a protein-protein interaction. 相似文献
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To study the function of GCR1, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Kluyveromyces lactis gene that complements the growth defect of a S. cerevisiae Deltagcr1 mutant was isolated. Introduction of this gene into the Deltagcr1 mutant also restored the activities of glycolytic enzymes. DNA sequencing of KlGCR1 predicted an open reading frame of a 767 amino acid protein with an overall identity of 33% and similarity of 48% to Gcr1p from S. cerevisiae. Its DDBJ/EMBL/GenBank Accession No. is AB046391. 相似文献
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对黄曲霉毒素B1(AFB1)降解菌株NMO-3 进行发酵培养基和培养条件优化,以期提高AFB1 降解率。方法:研究不同碳源、氮源、金属离子对AFB1 降解率的影响,最后选出最佳碳源、氮源和金属离子,通过正交回归试验,最后得出三者配方比。培养条件研究主要包括:初始pH 值、接种量、温度、种龄和降解时间等因素。结果:最终确定优化发酵培养基配方:果糖1.0%、胰蛋白胨1.0%、MgCl2 0.05mmol/L、其他发酵条件:起始pH 值为7.5、装液量25ml/300ml、接种量5%(V/V)、种子液培养时间为12h、控制降解温度为35℃、摇床转速140r/min、降解时间为72h。结论:经培养基成分和发酵参数的优化,AFB1 的降解率达到94.29%。 相似文献
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《Journal of dairy science》2022,105(2):1717-1730
Even though supplementations of essential AA (EAA) are often related to increased lactose yields in dairy cows, underlying mechanisms connecting EAA availability to the mammary glands and lactose synthesis are poorly understood. The objective of this study was to examine the effects of branched-chain AA (BCAA) including Leu, Ile, and Val on (1) glucose transporter (GLUT1) abundance and glucose uptake, (2) the abundance of proteins regulating lactose synthesis pathway, and (3) fractional synthesis rates of lactose (FSR) using bovine mammary epithelial cells (BMEC) and mammary tissues slices (MTS). The BMEC (n = 4) were allocated randomly to regular Dulbecco's Modified Eagle Medium with Ham's F12 (DMEM/F12) medium (+EAA) or +EAA deficient (by 90%) in all EAA (?EAA), all BCAA (?BCAA), only Leu (?Leu), only Ile (?Ile) or only Val (?Val). Western immunoblotting analyses, depletion of glucose in media, and a proteomic analysis were performed to determine the abundance of GLUT1 in the cell membrane, net glucose uptake, and the abundance of enzymes involved in lactose synthesis pathway in BMEC, respectively. The MTS (n = 6) were allocated randomly to DMEM/F12 medium having all EAA and 13C-glucose at concentrations similar to plasma concentrations of cows (+EAAp), and +EAAp deprived of all BCAA (?BCAAp) or only Leu (?Leup) for 3 h. The 13C enrichments of free glucose pool in MTS (EGlu-free) and the enrichments of glucose incorporated into lactose in MTS and media [ELactose-bound (T&M)] were determined and used in calculating FSR. In BMEC, ?BCAA increased the fraction of total GLUT1 translocated to the cell membrane and the fraction that was potentially glycosylated compared with +EAA. Among individual BCAA, only ?Leu was associated with a 63% increase in GLUT1 translocated to the cell membrane and a 40% increase in glucose uptake of BMEC. The ?BCAA tended to be related to a 75% increase in the abundance of hexokinase in BMEC. Deprivation of Leu tended to increase glucose uptake of MTS but did not affect EGlu-free, ELactose-bound (T&M), or FSR relative to +EAAp. On the other hand, ?BCAAp did not affect glucose uptake of MTS but was related to lower ELactose-bound (T&M), or FSR relative to +EAAp. Considering together, decreasing Leu supply to mammary tissues enhances GLUT1 and thus glucose uptake, which, however, does not affect lactose synthesis rates. Moreover, the deficiency of other BCAA, Ile, and Val alone or together with the deficiency of Leu seemed to decrease lactose synthesis rates without affecting glucose uptake. The data also emphasize the importance of addressing the effect of the supply of other nutrients to the mammary glands than the precursor supply in describing the synthesis of a milk component. 相似文献
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A mutant of Schwanniomyces castellii with reduced glucose phosphorylation and with practically no phosphorylation of fructose was investigated. Carbon catabolite represion of α-glucosidase and amylases was reduced. Repression of β-galactosidase was normal. We have compared in continuous culture this mutant strain with wild type and another previously described mutant. The relationship between the specific rate of glucose consumption (Qs) and residual glucose concentration (s) in an inverse mode, suggests that there may be two types of transport of glucose. Mutation at the phosphorylation level causes apparent modification of the kinetic parameters of glucose uptake rate. The consequence of mutation at the phosphorylation level on biomass production was discussed. 相似文献
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为研究脯氨酰羟化酶(prolylhydroxylase,PHD)对宰后牦牛肉低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、糖酵解及肉品质的影响,以牦牛背最长肌为研究对象,分析二甲基乙二酰氨基乙酸(dimethyloxaloylglycine,DMOG)处理组和生理盐水对照组中HIF-1α表达量、糖酵解关键酶活性、糖酵解程度、肉品质等指标的变化。结果显示,在宰后成熟过程中,HIF-1α表达量随成熟时间的延长呈先升高后降低的趋势,但由于DMOG的作用,HIF-1α表达量在宰后12 h内显著升高(P<0.05),说明抑制PHD活性可以上调HIF-1α表达量。己糖激酶(hexokinase,HK)活性、磷酸果糖激酶(phosphofructokinase,PFK)活性和丙酮酸激酶(pyruvate kinase,PK)活性均随成熟时间的延长呈先升高后降低的趋势,同时DMOG组的HK、PFK、PK的活性在3~120 h均显著高于对照组(P<0.05);DMOG组的糖原含量和pH值均在3~72 h显著低于对照组(P<0.05),而乳酸含量显著高于对照组(P<0.05),说明PHD介导HIF-1α上调糖酵解酶活性,加速糖酵解进程。同时,DMOG组的剪切力在宰后成熟3~72 h显著高于对照组(P<0.05),L*值在6~168 h显著高于对照组(P<0.05),而a*值在成熟过程中始终显著高于对照组(P<0.05);DMOG组的肌纤维直径和肌纤维面积均低于对照组,肌纤维间隙高于对照组,但差异不显著。说明PHD介导HIF-1α对宰后肉品质的形成产生影响,其中剪切力与L*值的变化由pH值变化引起,而a*值的变化原因需要进一步探究。结果表明,在宰后成熟过程中,PHD通过上调HIF-1α表达量进而增强糖酵解关键酶活性,加速糖酵解进程,引起肌肉内环境的变化,最终影响宰后肉品质的形成。 相似文献
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从青岛海藻化工厂海带浸泡液中分离得到KlebsiellaoxytocaXCH 1菌,研究了KlebsiellaoxytocaXCH 1菌产胞外多糖的摇瓶发酵情况,探讨了培养基中碳源、氮源、起始碳源质量浓度、碳氮比、温度、初始pH值、种龄、接种量、装液量等因素对KlebsiellaoxytocaXCH 1菌产生胞外多糖的影响.结果表明,最适宜条件为:以甘露醇为碳源,硫酸铵为氮源,起始甘露醇质量浓度为3g/dL,碳氮质量比为150∶1,温度28℃,初始pH值7~8,种龄54h,接种体积分数8%,250mL摇瓶装液量为40mL,并保持良好的供氧条件. 相似文献
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以绿熟期杏果为材料,分析了热风干制和自然晒制过程中己糖激酶、果糖激酶、蔗糖相关酶活性及糖含量的变化规律,旨在明确干制过程中关键酶调控糖代谢的作用机制。结果表明,果糖、葡萄糖、蔗糖占可溶性总糖的比例在干制前分别为33.52%、47.25%、45.22%,干制后分别平均为9.70%、28.24%、54.16%,果糖、葡萄糖和蔗糖呈显著负相关,因此干制条件下杏果中蔗糖的积累主要来源于己糖的转化。干制期间杏果中分解类酶活性(NI、SSs、AI)降低,且与果糖、葡萄糖呈正相关,分解类酶的调控以NI和AI为主。蔗糖合成类酶(SPS、SSs)在蔗糖代谢中起到辅助调控作用且热风40~50℃温度处理能有效地提高其活性,使蔗糖含量及蔗糖占可溶性总糖的比例增大。果糖激酶和己糖激酶也在干制前期略有升高,高活性的果糖激酶和己糖激酶利于增加果实中己糖的消耗,为蔗糖的积累提供基础。 相似文献
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为研究缺氧诱导因子(hypoxia-inducible factor 1,HIF-1)对滩羊肉宰后成熟48 h内糖酵解关键酶、能量水平、pH值及肉色的影响,本实验以4 ℃成熟2、6、12、24、48 h的滩羊背最长肌为研究对象,测定不同成熟期HIF-1表达量,磷酸丙糖异构酶(triose-phosphate isomerase,TPI)活力,能量物质三磷酸腺苷(adenosine triphosphate,ATP)、二磷酸腺苷(adenosine diphosphate,ADP)、单磷酸腺苷(adenosine monophosphate,AMP)水平,肌肉pH值及肉色的变化,并对HIF-1表达量与TPI活力,ATP、ADP、AMP含量,pH值及肉色进行相关性分析。结果表明,宰后2~24 h内HIF-1表达量迅速升至最高值,24 h后有所下降;TPI活力于宰后6 h升至最高,随后逐渐降低;宰后48 h内能量物质ATP、ADP、AMP水平均随成熟时间延长逐渐降低;宰后48 h内pH值显著降低(P<0.05);肉色指标L*、a*值及b*值均随成熟时间延长显著升高(P<0.05)。宰后成熟2~48 h内滩羊肉HIF-1表达量与TPI活力、能量物质水平、肉色及pH值均显著相关(P<0.05、P<0.01),可能是由于受宰后缺氧信号诱导,HIF-1表达量于宰后6 h内迅速升高,HIF-1复合体转移至细胞核与糖酵解基因结合,促进糖酵解相关基因表达,促使糖酵解途径增强,提高了糖酵解相关酶活力,并降低肌肉pH值。综上,HIF-1可能通过调控宰后糖酵解及肌肉pH值影响肉色。 相似文献
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不同年龄和部位羊肉中AMPK活性与糖酵解的差异 总被引:2,自引:0,他引:2
选取6月龄和18月龄宰后绵羊不同部位的骨骼肌,分别测定它们的AMPK活性和糖酵解各指标,研究不同年龄和部位羊肉中二者的差异,以探讨肌肉宰后生理的机理。结果表明:18月龄绵羊的AMPK活性极显著大于6月龄羊(P<0.01),pH下降幅度、肌糖原含量、乳酸含量和己糖激酶活性均极显著高于6月龄羊(P<0.01),乳酸脱氢酶活性显著高于6月龄羊(P<0.05)。并且同一年龄羊的各不同部位肌肉中AMPK活性和糖酵解也存在差异,运动量大的部位AMPK活性高,糖酵解速度快。因此得到结论,肌肉的AMPK活性较高时糖酵解速度也较快,反之较慢。 相似文献