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Transport Assays in Aspergillus nidulans
巢状曲霉转运蛋白动力学分析实验   

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Abstract

Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. These assays make use of A. nidulans germinating conidiospores, thus avoiding technical difficulties associated with the use of mycelia. The ease of construction genetic null mutants in this model fungus permits the rigorous characterization of any transporter in the absence of similar transporters with overlapping specificities, a common problem in relevant studies.

Keywords: Ascomycetes(子囊菌), Fungi(真菌), Kinetics(动力学), Specifcity(特异性), Transporter(转运)

Materials and Reagents

  1. p-aminobenzoic acid (Sigma-Aldrich, catalog number: P5669 )
  2. d-Biotin (Sigma-Aldrich, catalog number: B4501 )
  3. Calcium-D-pantothenate (Sigma-Aldrich, catalog number: 21210 )
  4. Riboflavine (Sigma-Aldrich, catalog number: R4500 )
  5. Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P9755 )
  6. KCl
  7. MgSO4.7H2O
  8. KH2PO4
  9. Na2B4O7.10H2O
  10. CuSO4.5H2O
  11. FeO4P.4H2O
  12. MnSO4.H2O
  13. Na2MoO4.2H2O
  14. ZnSO4.7H2O
  15. NaOH
  16. Tween 80 (Sigma-Aldrich, catalog number: P1754 )
  17. Radiolabelled substrate
    e.g. [8-3H]-xanthine, 22.8 Ci/mmol (Moravek Biochemicals, catalog number: MT537 )
    [2,8-3H]-hypoxanthine, 27.7 Ci/mmol (Moravek Biochemicals, catalog number: MT700 )
    [5-3H]-uracil, 23 Ci/mmol (Moravek Biochemicals, catalog number: MT610 )
  18. Non radiolabelled substrate
    e.g. Xanthine (Sigma-Aldrich, catalog number: X7375 )
    Hypoxanthine (Sigma-Aldrich, catalog number: H9377 )
    Uracil (Sigma-Aldrich, catalog number: U0750 )
  19. Toluol (AppliChem GmbH, catalog number: A3393 )
  20. Triton X-100
  21. 2,5-Diphenyloxazole (PPO) (Sigma-Aldrich, catalog number: D4630 )
  22. 1,4-bis (5-phenyloxazol-2-yl) benzene (POPOP) (Sigma-Aldrich, catalog number: P3754 )
  23. Complete Media (CM) (see Recipes)
  24. Minimal Media (MM) (see Recipes)
  25. Scintillation Fluid (see Recipes)

Equipment

  1. Petri dishes
  2. Neubauer counting-chamber slide
  3. Spatula
  4. Orbital shaking incubator
  5. Incubator at 37 °C
  6. Nylon net filter 60 μm (Merck KGaA, catalog number: NY60 )
  7. 50 ml Falcon tubes
  8. 1.5 ml centifuge tubes
  9. Centrifuge
  10. Vortex
  11. Scintillation vials
  12. Scintillation counter
  13. Heat block
  14. Magnetic stirrer
  15. Magnetic strirr bar
  16. pH meter
  17. Pasteur pipette

Software

  1. GraphPad Prism software (Amillis et al., 2004)

Procedure

  1. Inoculate a petri dish of CM with the strain of interest and let it reach full growth at 37 °C for 96 h.
  2. Using a spatula transfer one quarter of the fully grown colony (~4 cm) in a 50 ml Falcon tube containing 2 ml of 0.01% v/v Tween 80 in water. This amount usually corresponds to 108 conidiospores. The accurate amount of conidiospores can be estimated using a Neubauer counting-chamber slide or by measuring viable conidiospores after standard serial dilutions and plating on CM.
  3. Vortex well the sample for separating the conidiospores.
  4. Inoculate a 100 ml flask containing 25 ml MM supplemented with appropriate carbon (C) (e.g. Glucose 1% w/v) and nitrogen (N) (e.g. NaNO3 10 mM) sources and necessary vitamins (e.g. D-biotin 0.02 μg/ml) with the conidiospores filtered through a Nylon net filter 60 μΜ. (All necessary supplements and concentrations for A. nidulans strains can be found at www.fgsc.net.)
  5. Incubate for 3-5 h at 37 °C, shaking with 140 rpm, for the germinating conidiospores to reach a stage just before germ tube emergence. The time and temperature of incubation can change depending on the expression profile of the transporter of interest and the auxotrophic requirements of the strain. All transporters studied up to date in our lab (for example the purine/pyrimidine transporters UapA, UapC, AzgA, FcyB, FurD) reach maximum expression before germ tube emergence, driven by an unknown developmental control, irrespectively of the presence or absence of their substrates or other physiological conditions (Amillis et al., 2004; Vlanti and Diallinas, 2008; Amillis et al., 2007). In mycelia, transporter expression is very much dependent on physiological conditions (e.g. induction by substrates or/and N or C catabolite repression).
  6. While conidiospores germinate, prepare the stock solution of radiolabeled (usually 3H or 14C) substrate of interest in water or MM so that for each assay 25 μl of the stock will be used.
  7. Collect the conidiospores by centrifuging the culture in a 50 ml Falcon tube for 5 min, at 3,000 x g, room temperature.
  8. Discard the supernatant and resuspend the pellet in 5 ml standard MM.
  9. Distribute the spores in 75 μl aliquots in eppendorf tubes and use as many as needed. Conidiospore suspensions can be kept at 4 °C for at least 24 h without loss of transport activity.
  10. Incubate conidiospore aliquots at 37 °C in a heat block for 5 min before addition of radiolabeled substrate.
  11. Radiolabeled substrate is added for different periods of time. Most transporters show linearly increased activities for at least 1 min. For measuring initial uptake rates, which are necessary for determining Km and apparent Vmax values, the proper time of incubation must be defined for each transporter through a time-course experiment. For steady state substrate accumulation a period of 5 min is used. Usual time points are 10, 20, 30, 60 and 120 sec. For each time point, measurements are performed in triplicate. The temperature used for the incubation with the radiolabeled substrate depends on the transporter being studied at each experiment and the experiment being held, temperature for most experiments is 37 °C. The transport reaction is stopped by adding an equal volume (100 μl) of cold unlabeled substrate at 100-1,000 fold excess concentration, related to radiolabelled substrate, and direct transfer of the assay/eppendorf tube in an ice bucket.
  12. Centrifuge the samples at 11,000 x g for 3 min at 4 °C.
  13. Remove the supernatant through aspiration under vacuum using a Pasteur pipette. It is important to remove all the supernatant without losing any cells.
  14. Wash the pellet of cells once with 1 ml ice cold MM and centrifuge at 11,000 x g for 3 min. Remove the supernatant as before.
  15. Resuspend the pellet in 1 ml of scintillation fluid and put the eppendorf tubes into scintillation vials. Use a scintillation counter to measure substrate accumulation in the cells.
  16. Analysis of transport measurements is performed using GraphPad Prism software. Radioactive counts should be converted to substrate concentration/min/conidiospores, based on the concentration and specific activity of the stock of radioactive substrate used.
  17. For Km determination of a transporter, different substrate concentrations should be used, for a fixed incubation time, previously determined to reflect initial uptake rates. This is usually 1 min. The range of concentrations used is determined at first empirically. In the final experiment, at least three concentration points below and above the apparent Km value should be used. For each concentration point measurements are performed in triplicate.
  18. The stock solutions are prepared using a mixture of fixed radiolabeled substrate and increasing concentrations of non-radiolabeled substrate, so that for each assay 25 μl of the stock will be used.
  19. Terminate transport assays and perform measurements as described above.
  20. For Ki determination of a transporter, the method is identical to the one for Km determination, but stock solutions are prepared using a mixture of fixed radiolabeled substrate and increasing concentrations of non-radiolabeled putative inhibitors. For each concentration point measurements are performed in triplicate.
  21. Km and Vmax determination is carried out using typical Lineweaver-Burk plot analysis that is based on the Michaelis-Menten equation for enzyme kinetics V = Vmax[S]/(Km + [S]), where V is the reaction velocity (the reaction rate), Km is the Michaelis–Menten constant, Vmax is the maximum reaction velocity, and [S] is the substrate concentration. The Lineweaver-Burk plot depicts the linear expression of the previous equation which is transformed to the following: 1/V = (Km/Vmax).(1/[S]) + 1/Vmax. The data obtained by this experiment correspond to the apparent velocity of the transporter for each substrate concentration. Ki measurements are determined by estimating IC50 values (inhibitor concentration for obtaining 50% inhibition) of given substrate/inhibitor, using the formula Ki = IC50/1 + [S]/Km, where [S] is the fixed concentration of radiolabeled substrate used. Another way to analyse the data is by using the GraphPad Prism Software through a non linear regression curve fit and sigmoidal dose response analysis. The IC50 value corresponds to the Km/i of the transporter. Quality factors for the analysis result are: R2 which should be > 0.99 and the Hill co-efficient which should be approximately -1 for a transporter with one binding site.
  22. The method described can be modified and adapted for most filamentous fungi that produce asexual spores, e.g. A. fumigatus. (Goudela et al., 2008)

Recipes

  1. Complete Media (1 L)
    Vitamin solution from 100x stock solution* 10 ml
    Salt solution from 50x stock solution** 20 ml
    Glucose 10 g
    Peptone 2 g
    Yeast Extract 1 g
    Cas-amino- acids 1 g
    Agar 10 g
    Add water to 1 L final volume
    Adjust the pH to 6.8 using NaOH
    Autoclave for 20 min

    *Vitamin stock solution
    p-aminobenzoic acid 20 mg
    d-biotin 1 mg
    Calcium-D-pantothenate 50 mg
    Riboflavin 50 mg
    Pyridoxine 50 mg
    Add water to 1 L final volume

    **Salt stock solution
    KCl 26 g
    MgSO4.7H2O 26 g
    KH2PO4 76 g
    Trace elements 20x stock solution*** 50 ml
    Add water to 1 L final volume

    ***Trace elements stock solution
    Na2B4O7.10H2O 40 mg
    CuSO4.5H2O 400 mg
    FeO4P.4H2O 714 mg
    MnSO4.H2O 728 mg
    Na2MoO4.2H2O 800 mg
    ZnSO4.7H2O 8 mg
    Add water to 1 L final volume

  2. Minimal Media
    Salt solution from 50x stock solution* 20 ml
    Add water until 1 L final volume
    Adjust the pH to 6.8 using NaOH
    Autoclave for 20 min
  3. Scintillation Fluid (1 L)
    Toluol 666 ml
    PPO 2.66 g
    POPOP 0.0066 g
    2 h stirring in RT
    Add Triton-X 100 333 ml
    Overnight stirring

Acknowledgments

This protocol was adapted from the following publications: Diallinas et al. (1995); Koukaki et al. (2005); Meintanis et al. (2000); Tazebay et al. (1997). E.K. works in the laboratory of G.D, and is co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Thales, Investing in knowledge society through the European Social Fund.

References

  1. Amillis, S., Cecchetto, G., Sophianopoulou, V., Koukaki, M., Scazzocchio, C. and Diallinas, G. (2004). Transcription of purine transporter genes is activated during the isotropic growth phase of Aspergillus nidulans conidia. Mol Microbiol 52(1): 205-216.
  2. Amillis, S., Hamari, Z., Roumelioti, K., Scazzocchio, C. and Diallinas, G. (2007). Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans. Mol Membr Biol 24(3): 206-214.
  3. Diallinas, G., Gorfinkiel, L., Arst, H. N., Jr., Cecchetto, G. and Scazzocchio, C. (1995). Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes. J Biol Chem 270(15): 8610-8622.
  4. Goudela, S., Reichard, U., Amillis, S. and Diallinas, G. (2008). Characterization and kinetics of the major purine transporters in Aspergillus fumigatus. Fungal Genet Biol 45(4): 459-472. 
  5. GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA. www.graphpad.com.
  6. Koukaki, M., Vlanti, A., Goudela, S., Pantazopoulou, A., Gioule, H., Tournaviti, S. and Diallinas, G. (2005). The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway. J Mol Biol 350(3): 499-513.
  7. Meintanis, C., Karagouni, A. D. and Diallinas, G. (2000). Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family. Mol Membr Biol 17(1): 47-57.
  8. Tazebay, U. H., Sophianopoulou, V., Scazzocchio, C. and Diallinas, G. (1997). The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation. Mol Microbiol 24(1): 105-117.
  9. Vlanti, A. and Diallinas, G. (2008). The Aspergillus nidulans FcyB cytosine-purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis. Mol Microbiol 68(4): 959-977.

简介

运输测定允许通过测量表观上的最小值和最小值来测量特定转运蛋白的直接动力学分析,以及 允许通过竞争测定表征底物特异性谱。 在本协议中,我们描述了在模型丝状子囊菌实施吸收测定的快速和容易的方法 Aspergillus nidulans 。 这些测定利用了A。 构巢曲霉发芽分生孢子,从而避免与使用菌丝体相关的技术困难。 在该模型真菌中构建遗传无效突变体的容易性允许在不存在具有重叠特异性的类似转运蛋白的情况下严格表征任何转运蛋白,这是相关研究中的常见问题。

关键字:子囊菌, 真菌, 动力学, 特异性, 转运

材料和试剂

  1. 对氨基苯甲酸(Sigma-Aldrich,目录号:P5669)
  2. d-Biotin(Sigma-Aldrich,目录号:B4501)
  3. D-泛酸钙(Sigma-Aldrich,目录号:21210)
  4. 核黄素(Sigma-Aldrich,目录号:R4500)
  5. 盐酸吡哆醇(Sigma-Aldrich,目录号:P9755)
  6. KCl
  7. MgSO 4。 7H 2 O
  8. KH 2 PO 4
  9. lt; sub> 2 H< sub> 4< sub> 7< sub>
  10. CuSO 4 5H sub 2 O
  11. FeO 4 P 4H 2 O
  12. MnSO 4 H O
  13. Na 2 MoO 4 sub 。 2H O
  14. ZnSO 4 。 7H O
  15. NaOH
  16. 吐温80(Sigma-Aldrich,目录号:P1754)
  17. 放射性标记的底物
    例如22.8Ci/mmol(Moravek Biochemicals,目录号:MT537)[vi] [8-] 3 H] - 黄嘌呤,
    [2,8-三H] - 次黄嘌呤,27.7Ci/mmol(Moravek Biochemicals,目录号:MT700)
    [5-顺-3 H] - 尿嘧啶,23Ci/mmol(Moravek Biochemicals,目录号:MT610)
  18. 非放射性标记的底物
    例如黄嘌呤(Sigma-Aldrich,目录号:X7375)
    次黄嘌呤(Sigma-Aldrich,目录号:H9377)
    尿嘧啶(Sigma-Aldrich,目录号:U0750)
  19. Toluol(AppliChem GmbH,目录号:A3393)
  20. Triton X-100
  21. 2,5-二苯基恶唑(PPO)(Sigma-Aldrich,目录号:D4630)
  22. 1,4-双(5-苯基恶唑-2-基)苯(POPOP)(Sigma-Aldrich,目录号:P3754)
  23. 完成媒体(CM)(参见配方)
  24. 最小媒体(MM)(请参阅配方)
  25. 闪烁液(见配方)

设备

  1. 培养皿
  2. Neubauer计数室幻灯片
  3. 小铲
  4. 轨道振荡培养箱
  5. 37℃的培养箱
  6. 尼龙网过滤器60μm(Merck KGaA,目录号:NY60)
  7. 50ml Falcon管
  8. 1.5 ml Centifuge管
  9. 离心机
  10. 涡流
  11. 闪烁瓶
  12. 闪烁计数器
  13. 热块
  14. 磁力搅拌器
  15. 磁力杆
  16. pH计
  17. 巴斯德移液器

软件

  1. GraphPad Prism软件(Amillis等人,2004)

程序

  1. 用感兴趣的菌株接种CM的培养皿,并使其在37℃达到完全生长96小时。
  2. 使用刮刀在含有2ml的0.01%v/v吐温80的水的50ml Falcon管中转移四分之一完全生长的菌落(〜4cm)。该量通常对应于10 8个分生孢子。可以使用Neubauer计数室载玻片或通过在标准连续稀释和在CM上铺板后测量活的分生孢子来估计分生孢子的准确量。
  3. 涡旋好的样品分离分生孢子。
  4. 接种含有补充有合适的碳(C)(例如葡萄糖1%w/v)和氮气(N)(例如,NaNO 3)的25ml MM的100ml烧瓶, 3×10mM)来源和必需的维生素(例如d-生物素0.02μg/ml),通过尼龙网过滤器60μM过滤分生孢子。 (可在 www.fgsc.net 上找到 A.nidulans 菌株的所有必需的补充剂和浓度。)
  5. 孵育3-5小时,在37℃,以140 rpm摇动,发芽的分生孢子到达即将在胚芽管出现之前的阶段。孵育的时间和温度可以根据表达谱改变 的感兴趣的转运蛋白和该菌株的营养缺陷需求。在我们实验室研究的所有转运蛋白(例如嘌呤/嘧啶转运蛋白UapA,UapC,AzgA,FcyB,FurD)在胚芽管出现前达到最大表达,由未知的发育控制驱动,与其存在或不存在无关底物或其他生理条件(Amillis等人,2004; Vlanti和Diallinas,2008; Amillis等人,2007)。在菌丝体中,转运蛋白的表达非常依赖于生理条件(例如底物诱导或/和N或C分解代谢物抑制)。
  6. 当分生孢子萌发时,制备放射性标记的(通常为3 H或14 C)感兴趣的底物在水或MM中的储备溶液,使得对于每次测定,25μl的储备液使用。
  7. 通过将培养物在50ml Falcon管中在3,000xg,室温下离心5分钟收集分生孢子。
  8. 弃去上清液并将沉淀重悬在5ml标准MM中。
  9. 分配75微升等分试样在eppendorf管中的孢子,并使用所需的多。分生孢子悬浮液可以在4℃保持至少24小时,而不丧失转运活性。
  10. 在37℃下在加热块中孵育分生孢子等分试样5分钟,然后加入放射性标记的底物。
  11. 加入放射性标记的底物不同的时间。大多数转运蛋白显示线性增加的活动至少1分钟。为了测量确定 k m 和表观 max 值,必须通过时间过程实验为每个转运蛋白确定适当的孵育时间。对于稳态底物积累,使用5分钟的时间段。通常的时间点为10,20,30,60和120秒。对于每个时间点,一式三份进行测量。用于与放射性标记的底物孵育的温度取决于在每个实验中研究的转运蛋白并且保持实验,大多数实验的温度为37℃。通过加入与放射性标记的底物相关的等体积(100μl)100-1000倍过量浓度的冷的未标记底物,并在冰桶中直接转移测定/eppendorf管,停止转运反应。
  12. 在4℃下,将样品以11,000xg离心3分钟
  13. 通过使用巴斯德吸管在真空下抽吸去除上清液。重要的是清除所有的上清液,而不损失任何细胞
  14. 用1ml冰冷的MM洗涤细胞沉淀一次,并以11,000xg离心3分钟。如前所述除去上清液。
  15. 将沉淀重悬在1ml闪烁液中,将eppendorf管置于闪烁瓶中。使用闪烁计数器来测量细胞中的底物积累。
  16. 运输测量的分析使用GraphPad Prism软件进行。基于所用放射性底物的浓度和比活性,应将放射性计数转化为底物浓度/分钟/分生孢子。
  17. 对于转运体的 测定,应该使用不同的底物浓度,固定的孵育时间,这通常是1分钟。使用的浓度范围首先根据经验确定。在最后的实验中,应当使用低于和高于表观上的 值的至少三个浓度点。对于每个浓度点,测量一式三份
  18. 使用固定的放射性标记的底物和递增浓度的非放射性标记的底物的混合物制备储备溶液,使得对于每次测定将使用25μl的储备液。
  19. 终止运输检测并进行上述测量
  20. 对于转运器的 K i 确定,该方法与 K > m 测定,但是使用固定的放射性标记底物和递增浓度的非放射性标记的推定抑制剂的混合物制备储备溶液。对于每个浓度点,测量一式三份
  21. 使用基于用于酶动力学的Michaelis-Menten方程的典型的Lineweaver-Burk图分析,来测定酶的动力学 = sub> [S] /( K m + [S]),其中反应速率), m 是Michaelis-Menten常数, 是最大反应速度,[S]是底物浓度。 Lineweaver-Burk图描绘了先前等式的线性表达式,其转换为以下形式:1/V =( K m / sub> max 。通过该实验获得的数据对应于每种底物浓度的转运体的表观速度。通过使用公式估计IC 50值(用于获得50%抑制的抑制剂浓度)确定给定底物/抑制剂的Ki 测量值。 sub> i i = IC 50 /1 + [S]/其中[S]是使用的放射性标记底物的固定浓度。分析数据的另一种方式是通过使用GraphPad Prism软件通过非线性回归曲线拟合和S形剂量反应分析。 IC 50 值对应于运输商的 K m/i 。分析结果的质量因子是:R 2 其应该> 0.99和Hill系数,对于具有一个结合位点的运输者,该系数应该约为-1
  22. 所描述的方法可以修改和适用于产生无性孢子的大多数丝状真菌,例如。烟曲霉。 (Goudela et al。,2008)

食谱

  1. 完成媒体(1 L)
    来自100x储备溶液的维生素溶液* 10ml
    盐溶液从50x储备溶液** 20 ml
    葡萄糖10克
    蛋白胨2 g
    酵母提取物1 g
    Cas氨基酸1 g
    琼脂10克
    加水至1 L最终体积
    用NaOH
    调节pH至6.8 高压灭菌20分钟

    *维生素储液
    对氨基苯甲酸20mg
    d生物素1mg
    D-泛酸钙50mg
    核黄素50 mg
    吡哆醇50mg
    加水至1 L最终体积

    **盐储液
    KCl 26 g
    MgSO 4。 7H 2 O 26g



    微量元素20x储备溶液*** 50 ml
    加水至1 L最终体积

    ***微量元素储备液
    Na 2 O 2 b 4 O 7 sub 10 H 2 O 40mg
    > CuSO 4 5H O 400 mg

    P 4H 2 714mg
    lt; sub> 4< sup> H 728mg
    800 mg
    ZnSO 4 。 7H 2 O 8 mg
    加水至1 L最终体积

  2. 最小媒体
    盐溶液从50x储备溶液* 20ml
    加水至1 L最终体积
    用NaOH
    调节pH至6.8 高压灭菌20分钟
  3. 闪烁液(1 L)
    Toluol 666 ml
    PPO 2.66克
    POPOP 0.0066克
    在RT下搅拌2小时
    加入Triton-X 100 333 ml
    过夜搅拌

致谢

该协议改编自以下出版物:Diallinas等人(1995); Koukaki等人(2005); Meintanis等人(2000); Tazebay等人(1997)。英国在GD的实验室工作,由欧盟(欧洲社会基金ESF)和希腊国家基金通过国家战略参考框架(NSRF)的"教育和终身学习"运作计划 - 研究资助计划:Thales,通过欧洲社会基金投资知识社会。

参考文献

  1. Amillis,S.,Cecchetto,G.,Sophianopoulou,V.,Koukaki,M.,Scazzocchio,C.and Diallinas,G。(2004)。 嘌呤转运蛋白基因的转录在构巢曲霉的各向同性生长期期间被激活 conidia。 Mol Microbiol 52(1):205-216。
  2. Amillis,S.,Hamari,Z.,Roumelioti,K.,Scazzocchio,C。和Diallinas,G。(2007)。 表达调节和尿嘧啶转运蛋白在构巢曲霉中的底物相互作用的动力学模拟 em> 。 Mol Membr Biol 24(3):206-214
  3. Diallinas,G.,Gorfinkiel,L.,Arst,H.N.,Jr.,Cecchetto,G。和Scazzocchio,C。(1995)。 编码基因的基因的遗传和分子表征 广泛的特异性嘌呤通透酶 揭示了在原核生物和真核生物中保守的转运蛋白的新家族 270(15):8610-8622。 br />
  4. Goudela,S.,Reichard,U.,Amillis,S.and Diallinas,G。(2008)。 烟曲霉中主要嘌呤转运蛋白的表征和动力学。真菌基因Biol 45(4):459-472。 
  5. 用于Windows的GraphPad Prism版本5.00,GraphPad Software,San Diego California USA。 www.graphpad.com
  6. Koukaki,M.,Vlanti,A.,Goudela,S.,Pantazopoulou,A.,Gioule,H.,Tournaviti,S.and Diallinas,G。(2005)。 UapA中的核碱基 - 抗坏血酸转运蛋白(NAT)特征基序定义了嘌呤易位途径的功能。 J Mol Biol 350(3):499-513
  7. Meintanis,C.,Karagouni,A.D.and Diallinas,G。(2000)。 氨基酸残基N450和Q449对于UapA的吸收能力和特异性至关重要,UapA是一种核碱基 - 抗坏血酸转运蛋白家族。 Mol Membr Biol 17(1):47-57。
  8. Tazebay,U.H.,Sophianopoulou,V.,Scazzocchio,C。和Diallinas,G。(1997)。 编码曲霉菌的主要脯氨酸转运蛋白的基因 构巢曲霉在分生孢子萌发期间和在脯氨酸诱导和氨基酸饥饿反应中上调。 Mol Microbiol 24(1):105-117。
  9. Vlanti,A。和Diallinas,G。(2008)。 构巢曲霉 FcyB胞嘧啶 - 嘌呤清除剂在萌发期间高度表达, 在生殖室中并通过内吞作用下调。 Mol Microbiol 68(4):959-977。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Krypotou, E. and Diallinas, G. (2013). Transport Assays in Aspergillus nidulans. Bio-protocol 3(22): e971. DOI: 10.21769/BioProtoc.971.
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