Name: concanavalin a-based sedimentation assay to measure substrate binding of glucan phosphatases
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这项研究得到了美国国家科学基金会奖MCB-2012074的支持。作者感谢佛罗里达大学生物化学与分子生物学系的Craig W. Vander Kooi博士的宝贵讨论和支持。作者还感谢佛罗里达大学生物化学和分子生物学系的Matthew S. Gentry博士的支持。我们要感谢斯基德莫尔学院神经科学项目主席Sara Lagalwar博士允许我们使用LICOR C数字印迹扫描仪进行蛋白质印迹成像。

1. ConA-琼脂糖珠的制备
<ol><li>制备 250 mL 含有 67 mM HEPES （pH 7.5）、10 mM MgCl 2 和 0.2 mM CaCl2 的结合缓冲液。使用1M NaOH溶液调节pH值。</li>
	<li>将 250 μL ConA-琼脂糖珠悬浮液移液到 1.5 mL 微量离心管中。将内容物在4°C下以10,000× g 离心30秒。 弃去上清液。 注意：用于测定的每个支链淀粉浓度需要 250 μL 1.5 mL 微量离心管中的 ConA-琼脂糖珠。</li>
	<li>向含有 250 μL ConA-...

<ol>
	<li>Meekins, D. A., Vander Kooi, C. W., Gentry, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanisms+of+plant+glucan+phosphatases+in+starch+metabolism.">Structural mechanisms of plant glucan phosphatases in starch metabolism.</a> The FEBS Journal. 283 (13), 2427-2447 (2016).</li><li>Gentry, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phosphatase+laforin+crosses+evolutionary+boundaries+and+links+carbohydrate+metabolism+to+neuronal+disease.">The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease.</a> The Journal of Cell Biology. 178 (3), 477-488 (2007).</li><li>Worby, C. A., Gentry, M. S., Dixon, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laforin,+a+dual+specificity+phosphatase+that+dephosphorylates+complex+carbohydrates.">Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates.</a> The Journal of Biological Chemistry. 281 (41), 30412-30418 (2006).</li><li>Gentry, M. S., Pace, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+the+glucan+phosphatase+laforin+is+linked+to+rates+of+molecular+evolution+and+the+glucan+metabolism+of+the+organism.">Conservation of the glucan phosphatase laforin is linked to rates of molecular evolution and the glucan metabolism of the organism.</a> BMC Evolutionary Biology. 9, 138 (2009).</li><li>Niittyla, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similar+protein+phosphatases+control+starch+metabolism+in+plants+and+glycogen+metabolism+in+mammals.">Similar protein phosphatases control starch metabolism in plants and glycogen metabolism in mammals.</a> The Journal of Biological Chemistry. 281 (17), 11815-11818 (2006).</li><li>Kotting, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STARCH-EXCESS4+is+a+laforin-like+Phosphoglucan+phosphatase+required+for+starch+degradation+in+Arabidopsis+thaliana.">STARCH-EXCESS4 is a laforin-like Phosphoglucan phosphatase required for starch degradation in Arabidopsis thaliana.</a> The Plant Cell. 21 (1), 334-346 (2009).</li><li>Comparot-Moss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+phosphatase,+LSF1,+is+required+for+normal+starch+turnover+in+Arabidopsis+leaves.">A putative phosphatase, LSF1, is required for normal starch turnover in Arabidopsis leaves.</a> Plant Physiology. 152 (2), 685-697 (2010).</li><li>Zeeman, S. C., Northrop, F., Smith, A. M., Rees, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+starch-accumulating+mutant+of+Arabidopsis+thaliana+deficient+in+a+chloroplastic+starch-hydrolysing+enzyme.">A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme.</a> The Plant Journal: For Cell and Molecular Biology. 15 (3), 357-365 (1998).</li><li>Kotting, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+enzyme+required+for+starch+metabolism+in+Arabidopsis+leaves.+The+phosphoglucan,+water+dikinase.">Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The phosphoglucan, water dikinase.</a> Plant Physiology. 137 (1), 242-252 (2005).</li><li>Tagliabracci, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laforin+is+a+glycogen+phosphatase,+deficiency+of+which+leads+to+elevated+phosphorylation+of+glycogen+in+vivo.">Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo.</a> Proceedings of the National Academy of Sciences. 104 (49), 19262-19266 (2007).</li><li>Gentry, M. S., Guinovart, J. J., Minassian, B. A., Roach, P. J., Serratosa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lafora+disease+offers+a+unique+window+into+neuronal+glycogen+metabolism.">Lafora disease offers a unique window into neuronal glycogen metabolism.</a> The Journal of Biological Chemistry. 293 (19), 7117-7125 (2018).</li><li>Brewer, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+pathogenic+lafora+bodies+in+lafora+disease+using+an+antibody-enzyme+fusion.">Targeting pathogenic lafora bodies in lafora disease using an antibody-enzyme fusion.</a> Cell Metabolism. 30 (4), 689-705 (2019).</li><li>Santelia, D., Zeeman, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+Arabidopsis+starch+research+and+potential+biotechnological+applications.">Progress in Arabidopsis starch research and potential biotechnological applications.</a> Current Opinion in Biotechnology. 22 (2), 271-280 (2011).</li><li>Raththagala, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanism+of+laforin+function+in+glycogen+dephosphorylation+and+lafora+disease.">Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease.</a> Molecular Cell. 57 (2), 261-272 (2015).</li><li>Meekins, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoglucan-bound+structure+of+starch+phosphatase+Starch+Excess4+reveals+the+mechanism+for+C6+specificity.">Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity.</a> Proceedings of the National Academy of Sciences. 111 (20), 7272-7277 (2014).</li><li>Vander Kooi, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+glucan+phosphatase+activity+of+Starch+Excess4.">Structural basis for the glucan phosphatase activity of Starch Excess4.</a> Proceedings of the National Academy of Sciences. 107 (35), 15379-15384 (2010).</li><li>Meekins, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Arabidopsis+glucan+phosphatase+like+sex+four2+reveals+a+unique+mechanism+for+starch+dephosphorylation.">Structure of the Arabidopsis glucan phosphatase like sex four2 reveals a unique mechanism for starch dephosphorylation.</a> The Plant Cell. 25 (6), 2302-2314 (2013).</li><li>Smith, A. M., Zeeman, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch:+A+flexible,+adaptable+carbon+store+coupled+to+plant+growth.">Starch: A flexible, adaptable carbon store coupled to plant growth.</a> Annual Review of Plant Biology. 71, 217-245 (2020).</li><li>Jane, J., Kasemuwan, T., Chen, J. F., Juliano, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+in+rice+and+other+starches.">Phosphorus in rice and other starches.</a> Cereal Foods World. 41 (11), 827-832 (1996).</li><li>Mak, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+kinetics+of+the+glucan+phosphatase+starch+excess4.">Cooperative kinetics of the glucan phosphatase starch excess4.</a> Biochemistry. 60 (31), 2425-2435 (2021).</li><li>Campbell, K. P., MacLennan, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+the+53,000-dalton+glycoprotein+from+the+sarcoplasmic+reticulum.">Purification and characterization of the 53,000-dalton glycoprotein from the sarcoplasmic reticulum.</a> The Journal of Biological Chemistry. 256 (9), 4626-4632 (1981).</li><li>Campbell, K. P., MacLennan, D. H., Jorgensen, A. O., Mintzer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+calsequestrin+from+canine+cardiac+sarcoplasmic+reticulum+and+identification+of+the+53,000+dalton+glycoprotein.">Purification and characterization of calsequestrin from canine cardiac sarcoplasmic reticulum and identification of the 53,000 dalton glycoprotein.</a> The Journal of Biological Chemistry. 258 (2), 1197-1204 (1983).</li><li>Davey, M. W., Sulkowski, E., Carter, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+human+fibroblast+interferon+to+concanavalin+A-agarose.+Involvement+of+carbohydrate+recognition+and+hydrophobic+interaction.">Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.</a> Biochemistry. 15 (3), 704-713 (1976).</li><li>Meekins, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+glucan+phosphatase+activity+against+polyglucan+substrates.">Mechanistic insights into glucan phosphatase activity against polyglucan substrates.</a> The Journal of Biological Chemistry. 290 (38), 23361-23370 (2015).</li><li>Wilkens, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+&#945;-glucan+phosphatases+SEX4+and+LSF2+display+different+affinity+for+amylopectin+and+amylose.">Plant &#945;-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose.</a> FEBS Letters. 590 (1), 118-128 (2016).</li><li>Atanasova, M., Bagdonas, H., Agirre, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+glycobiology+in+the+age+of+electron+cryo-microscopy.">Structural glycobiology in the age of electron cryo-microscopy.</a> Current Opinion in Structural Biology. 62, 70-78 (2020).</li><li>Doyle, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+binding+interactions+by+isothermal+titration+calorimetry.">Characterization of binding interactions by isothermal titration calorimetry.</a> Current Opinion in Biotechnology. 8 (1), 31-35 (1997).</li></ol>

这项研究证明了一种新的 体外 沉降测定法的成功开发，该测定法可以测定葡聚糖-葡聚糖磷酸酶相互作用的结合亲和力。该测定设计利用凝集素ConA通过葡萄糖的羟基残基 与 葡聚糖的特异性结合，间接捕获溶解的碳水化合物底物到琼脂糖珠上。这允许 通过离心分离 结合和未结合的蛋白质级分，并确定可溶的葡聚糖底物和葡聚糖磷酸酶的结合亲和力。发现所有测试的葡聚糖磷?...

葡聚糖磷酸酶是蛋白酪氨酸磷酸酶 （PTP） 超家族1 中功能多样化的双重特异性磷酸酶 （DSP） 亚家族的成员。它们存在于大多数生命形式中，包括广泛不同的光合生物、人类、脊椎动物以及一些无脊椎动物和原生生物2,3,4。植物含有三种已知的葡聚糖磷酸酶：淀粉过量4（SEX4），Like Four1（LSF1）和Like Four2（LSF2）5,6,7。缺乏葡聚糖磷酸酶的植物显示出暂时淀粉降解和淀粉在叶片中积累的速率降低8,9。拉福林是葡聚糖磷酸酶家族的创始成员，该家族使脊椎动物和人类的糖原去磷酸化3,10。拉福林的突变导致神经退行性拉福拉病，这是一种致命的常染色体隐性癫痫形式11。葡聚糖磷酸酶是糖原和淀粉代谢所必需的，并且已成为调节植物淀粉含量和治疗神经退行性Lafora病的重要酶12,13。最近对葡聚糖磷酸酶的X射线晶体学研究与模型葡聚糖底物揭示了底物结合和葡聚糖去磷酸化的催化机制14,15,16,17。然而，目前对葡聚糖磷酸酶如何与其生理底物结合的理解是不完整的。
淀粉是由80%-90%支链淀粉和10%-20%直链淀粉18制成的葡萄糖不溶性聚合物。植物葡聚糖磷酸酶的底物是磷酸化的碳水化合物分子，如糖原和淀粉颗粒。磷酸化的葡萄糖基残基以1：600的磷酸：葡萄糖基残基比例存在。有趣的是，磷酸盐仅存在于支链淀粉分子19上。主要植物葡聚糖磷酸酶SEX4作用于淀粉颗粒以去磷酸化支链淀粉分子。SEX4的X射线晶体结构与结构引导的诱变研究相结合，证明了SEX4对葡聚糖结构内不同位置的独特底物特异性15。我们最近表明，SEX4的生物学相关活性只能在作用于其可溶的支链淀粉底物20时观察到。然而，由于底物的结构复杂性、更广泛的结合特异性以及蛋白质与其底物之间的低结合亲和力，理解葡聚糖-SEX4 相互作用已被证明是困难的。这些问题阻碍了利用蛋白质-配体相互作用中常用的方法的能力，例如等温滴定量热法（ITC），核磁共振（NMR）波谱和基于酶联免疫吸附测定（ELISA）的测定。
有趣的是，我们对碳水化合物 - 蛋白质相互作用的大部分理解都来自研究凝集素。刀豆球蛋白A（ConA）是一种豆科凝集素家族，最初是从杰克豆中提取的蛋白质。ConA以高特异性结合碳水化合物，这有利于其在药物靶向和递送应用中的使用。ConA与含有非还原性α-D-甘露糖基和α-D-葡萄糖基的各种底物的结合已被广泛研究19,20。市售ConA结合琼脂糖珠通常用于纯化糖蛋白和糖脂21。ConA通过葡萄糖残基的C3，C4和C6羟基与这些葡聚糖结合。ConA-琼脂糖珠也已成功用于测量糖原-蛋白质和淀粉-蛋白质相互作用的结合22,23。在这项研究中，我们使用ConA-琼脂糖珠开发了一种结合测定法，以测量葡聚糖磷酸酶-支链淀粉相互作用的结合特异性。
以前，使用基于ConA的沉降测定来评估葡聚糖磷酸酶底物结合能力14,20,24。在这项研究中，使用相同的策略开发了一种确定葡聚糖-葡聚糖磷酸酶和碳水化合物相互作用的结合亲和力的新方法。该方法在研究各种可溶的碳水化合物-蛋白质相互作用方面也具有优势。

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	<tbody>
		<tr>
			<td>6x-His Tag monoclonal antibody (HIS.H8), HRP</td>
			<td>Therm Fisher Scientific</td>
			<td>MA1-21315-HRP</td>
			<td></td>
		</tr>
		<tr>
			<td>Biorad gel electrophoresis and Western blot kit</td>
			<td>Biorad&#160;</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208291</td>
			<td></td>
		</tr>
		<tr>
			<td>C-Digit blot scanner</td>
			<td>LICOR</td>
			<td>3600-00</td>
			<td>Blot scanner</td>
		</tr>
		<tr>
			<td>Complete protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin A-sepharose beads</td>
			<td>Sigma-Aldrich</td>
			<td>C9017</td>
			<td>This product contains&#160; in 0.1 M acetate buffer, pH 6, containing 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2 in 20% ethanol&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>5425R</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-5</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0 software</td>
			<td>GraphPad&#160;</td>
			<td>Version 8.0</td>
			<td>Data analysis software&#160;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H8651</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Studio</td>
			<td>LICOR</td>
			<td>3600-501</td>
			<td>Acquisition Software</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Fisher Scientific</td>
			<td>PI28312</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato amylopectin</td>
			<td>Sigma-Aldrich</td>
			<td>A8515</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast SDSPAGE Gels</td>
			<td>Genscript</td>
			<td>M00653S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>MP1TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Westernsure premium chemiluminescence substrate&#160;</td>
			<td>LI-COR</td>
			<td>&#160;926-95000</td>
			<td></td>
		</tr>
	</tbody>
</table>

concanavalin a-based sedimentation assay to measure substrate binding of glucan phosphatases

葡聚糖磷酸酶属于更大的双重特异性磷酸酶（DSP）家族，可去磷酸化葡聚糖底物，例如动物中的糖原和植物中的淀粉。具有模型葡聚糖底物的葡聚糖磷酸酶的晶体结构揭示了由DSP和碳水化合物结合结构域组成的独特的葡聚糖结合界面。然而，定量测量葡聚糖-葡聚糖磷酸酶与生理相关底物的相互作用对于葡聚糖磷酸酶家族酶的生物学理解和能量代谢的调节至关重要。本手稿报告了一种基于刀豆球蛋白 A （ConA） 的体外沉降测定，旨在检测葡聚糖磷酸酶对不同葡聚糖底物的底物结合亲和力。作为概念验证，测定了葡聚糖磷酸酶拟南芥淀粉过量4（SEX4）和支链淀粉的解离常数（KD）。SEX4突变体和葡聚糖磷酸酶家族的其他成员的表征进一步证明了该测定法在评估蛋白质 - 碳水化合物相互作用的差异结合方面的实用性。这些数据证明了该测定法适用于表征各种淀粉和糖原相互作用的蛋白质。

葡聚糖磷酸酶家族蛋白质的关键特征之一是它们与葡聚糖底物结合的能力。首先，使用SDS-PAGE分析SEX4与ConA-琼脂糖：支链淀粉珠的结合能力（图2A）。牛血清白蛋白（BSA）作为阴性对照，用于检测蛋白质与ConA-琼脂糖：支链淀粉珠的任何非特异性结合。蛋白质的SDS-PAGE分析显示沉淀级分中存在SEX4蛋白，上清液级分中存在BSA。W278A是一种已知的SEX4突变体，具有显着降低的葡聚糖?...

Watch this Scientific Journal Video about Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases at JoVE.com

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

该方法描述了一种基于凝集素的 体外 沉降测定，以量化葡聚糖磷酸酶和支链淀粉的结合亲和力。该共沉降测定法对于测量葡聚糖磷酸酶底物结合是可靠的，可应用于各种可溶的葡聚糖底物。

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

这种基于刀豆球蛋白A的体外沉降测定旨在测量葡聚糖磷酸酶对不同STA底物的结合亲和力。我们从这种方法中获得的信息对于我们理解葡聚糖磷酸酶家族至关重要。碳水化合物蛋白相互作用相对较弱，因此目前的方法无法检测葡聚糖磷酸酶和葡聚糖相互作用。我们基于凝集素的共沉降测定可以检测毫摩尔范围内的结合亲和力。SEX4突变体以及葡聚糖磷酸酶家族的其他成员的表征证明了该测定定量蛋白质碳水化合物相互作用的能力。测定中最具挑战性的部分之一是优化底物的浓度范围。我们建议从更广泛的范围开始以获得想法，然后缩小特定范围。首先，将250微升ConA琼脂糖珠悬浮液移入1.5毫升微量离心管中。将内容物在10， 000 G下在4摄氏度下离心30秒。弃去上清液后，向含有250微升ConA琼脂糖珠的每个管中加入750微升结合缓冲液。将试管在10， 000 G下在4摄氏度下离心一分钟，然后除去上清液。接下来，制备两倍稀释的溶解支链淀粉溶液，制成一系列两毫升稀释的支链淀粉溶液。为了制备ConA琼脂糖支链淀粉珠，将250微升每种稀释的支链淀粉溶液加入含有250微升ConA琼脂糖珠预平衡和结合缓冲液的管中。充分混合内容物，并用相应的支链淀粉浓度标记试管。将试管内容物在4摄氏度的旋转轮上孵育30分钟，并以10， 000G离心管1分钟。将 250 微升 ConA-琼脂糖支链淀粉珠与 100 微升含有 10 微克淀粉过量 4 或 SEX4 蛋白、10 毫摩尔二硫苏糖醇和 10 微摩尔蛋白酶抑制剂混合物的结合缓冲液混合。将内容物在 4 摄氏度下轻轻旋转孵育 45 分钟。然后将试管以10， 000G离心一分钟，并小心地将50微升上清液移入新的1.5毫升微量离心管中。向含有50微升收集的上清液级分的每个管中加入20微升4X SDS页面染料和10微升蒸馏水，并将其标记为S.同样，将20微升4X SDS页面染料和80微升蒸馏水加入含有洗涤的ConA琼脂糖支链淀粉SEX4珠子的管中。加入染料和水后，将所有样品在 95 摄氏度下加热 10 分钟。将ConA-琼脂糖支链淀粉SEX4珠以10， 000 G离心一分钟后，保存上清液并将其标记为P.Load 40微升标记为S的未结合蛋白质样品从最低底物浓度到最高底物浓度到4至12%预制聚丙烯酰胺凝胶孔中，为蛋白质分子量标记物保留第一泳道。使用第二块凝胶加载标记为P.的葡聚糖结合蛋白样品，将新鲜制备的SDS页电泳缓冲液添加到设备的两个腔室中，并以150伏电泳凝胶35分钟或直到染料前沿到达凝胶底部。然后从仪器中取出凝胶盒并取出垫片和玻璃板以分离凝胶以进行蛋白质印迹分析。制作一升转膜缓冲液，含有 5.8 克 tris 碱、2.9 克甘氨酸、0.37 克 SDS 和 200 毫升甲醇。要将大小分离的蛋白质从聚丙烯酰胺凝胶转移到硝酸纤维素膜上，请根据蛋白质免疫印迹转印方案组装海绵、滤纸、凝胶和硝酸纤维素膜，并在 70 伏下运行一小时。运行凝胶后，将硝酸纤维素膜在50毫升TBST缓冲液中1至5%牛血清白蛋白或乳蛋白的蛋白质溶液中孵育一小时，以防止非特异性蛋白质结合。使用TBST缓冲液洗涤膜三次，以除去任何未结合的封闭溶液。接下来，将膜与组氨酸标记蛋白特异性稀释的辣根过氧化物酶连接抗体孵育一小时。然后在TBST缓冲液中洗涤膜三次，以去除任何未结合的抗体。对于数字成像，将等份的化学发光底物溶液制成溶液，每个750微升在1.5毫升管中，并将膜在溶液中孵育至少五分钟。将膜蛋白面朝下放在印迹扫描仪上，然后运行采集软件以定量沉淀和上清液级分中的蛋白质。要获取数据，请使用带有印迹扫描仪的采集软件开始定量信号测量。将上清液和沉淀级分中的所有定量测量标准化为上样的总蛋白质。在饱和结合实验中，绘制蛋白质结合与支链淀粉浓度的百分比，并使用数据分析软件拟合数据以计算解离常数Kd.使用SDS页分析SEX4与ConA-琼脂糖支链淀粉珠的结合能力。注意到沉淀级分中存在SEX4蛋白，上清液级分中存在牛血清白蛋白。W278A是一种已知的SEX4突变体，具有显着降低的葡聚糖结合能力，出现在上清液级分中，表明该测定的效用和特异性。蛋白质印迹分析显示SEX4的检测显著增加，该方法特异性地用于检测具有末端组氨酸标签的葡聚糖磷酸盐。共沉降测定在三种不同的SEX4浓度下进行测试。虽然2微克SEX4足以通过化学发光检测进行可视化，但使用5或10微克SEX4可以进行更准确的检测。此外，注意到SEX4结合增加与支链淀粉浓度增加。测定支链淀粉和SEX4野生型蛋白在不同浓度下的结合亲和力，并将蛋白质与特定位点结合模型的结合百分比数据给出的Kd约为1.03毫克/毫升。评估该方法测量马铃薯淀粉、LSF2、玉米SEX4和马铃薯SEX4与马铃薯支链淀粉结合的适用性。结果表明，来自农艺重要作物的测试蛋白质也与支链淀粉不同结合。记住沉淀和上清液级分中所有定量测量值与总上样蛋白的标准化至关重要。为确保所有支链淀粉都与磁珠结合，请保存上清液级分以便稍后进行深层葡萄糖测定。葡聚糖磷酸酶采用不同的机制来结合、定位和去磷酸化碳水化合物。该方法允许探索葡聚糖磷酸酶与大型聚合物复合物中不同结构域的结合，以实现位置特异性去磷酸化。

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Measuring Protein Binding to F-actin by Co-sedimentation

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Quantitative Immunofluorescence to Measure Global Localized Translation

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...