Name: mapping metabolism: monitoring lactate dehydrogenase activity directly in tissue
Uploaded: 2018-06-21T14:00:00
Description: Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

文森特卡塞尔是丽塔. 艾伦基金会 (http://www.ritaallenfoundation.org) 的弥尔顿 e。这项工作由国家普通医学研究所 R01 GM081686、R01 AR070245、国立医学科学院 R01 GM0866465、Eli & 伊蒂丝广泛的再生医学 & 干细胞研究中心的赠款资助 (玫瑰丘陵和哈尔 Gaba 奖), 虹膜康托妇女健康中心/加州大学洛杉矶分校 CTSI NIH 补助金 UL1TR000124, 白血病淋巴瘤协会, 冲击奖从琼森综合癌症中心到和醋酸。在这份出版物中报告的研究得到了国家卫生研究院国家癌症研究所的支持, P50CA092131 奖号。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。醋酸是伊莱 & 伊蒂丝广泛的再生医学 & 干细胞研究中心的成员, 加州大学洛杉矶分校分子生物学研究所, 加州大学洛杉矶分校生物信息学部门计划。

所描述的所有实验都是由洛杉矶加利福尼亚大学动物保育委员会批准的。
1. 生成冷冻鼠标皮肤的幻灯片
<ol><li>弄死小鼠按照制度政策。 注意: 遵循保护服装的制度政策。所有涉及动物的议定书都必须得到机构动物保育委员会的批准。</li>
	<li>用动物发型剪掉老鼠的头发。</li>
	<li>用剪刀在皮肤上做切口。使用镊子, 把皮肤从老鼠身上抬开。用剪刀剪掉皮肤部分。</li>
	<li>用?...

<ol>
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这里描述的方法可以用来监测乳酸脱氢酶或其他代谢酶的活性, 产生 NADH 或, 在不同的细胞类型组织内或组织内的不同部分, 随着时间的推移。乳酸脱氢酶是了解干细胞和肿瘤生物学的重要酶, 在单个细胞内监测乳酸脱氢酶活性的能力很可能对这种酶的功能有重要的洞察力。
与其他方法相比, 本议定书的一个重要优点是, 监测酶活性的能力, 而不仅仅是蛋白质丰度, 可以导致更确?...

了解组织内酶具有高或低活性的部位, 对于理解发育和生理学是必不可少的。转录或蛋白质水平经常被用作酶活性的替代。虽然这类研究可以提供信息, 但它们并不能为确定酶的活性 (如平移后的修饰、蛋白质复合物的存在或酶的亚细胞定位) 的关键。当酶活性被直接测量, 它经常被监测在匀质蛋白裂解物, 不再包含有关单个细胞的信息, 或在组织内的高或低活动细胞的空间分布。
我们提供了一个详细的协议, 用于映射组织样本中酶活性的空间分布。该方法的基础是早期的研究表明, 四氮唑盐可用于本地化的活动, 脱氢酶, 硝酸和氧化酶在冷冻组织1。利用这些方法, 当质子转移到四氮唑盐2、3时, 就形成了水不溶 formazan。Glucose-6-phosphate 脱氢酶产生的四氮唑和质子, 并已检测到的活动。Glucose-6-phosphate 脱氢酶已监测在欧洲比目鱼肝细胞4, 在肺泡型2细胞的肺5和肾单位的肾脏5。四氮唑盐也被用来监测冷冻组织6中的 transketolase 活性。最近还使用类似的方法来监测相邻幻灯片7上同一组织中多个脱氢酶的活动。
这里我们描述了一种使用四氮唑盐监测乳酸脱氢酶活性的空间分布的方法 (图 1)。乳酸脱氢酶可将糖酵解生成的丙酮酸转化为乳酸, 并逆转反应。乳酸脱氢酶活性是丙酮酸盐进入 tricarboxylic 酸循环的重要决定因素, 与乳酸的分泌有关。血液中的乳酸水平经常被用来诊断一系列疾病, 包括癌症8、9、10, 因为它可以表明疾病或伤害已经损坏的细胞和酶已经释放。
乳酸脱氢酶基因有四: LDHA、LDHB、LDHC 和 LDHD11。LDHA 和 LDHB 被认为是来自早期 LDHA 基因12的重复。LDH 是活跃的聚丙烯和 LDHA 和 LDHB 可以形成 homotetramers 和 heterotetramers 彼此。据报道, LDHA 对丙酮酸有较高的亲和力, 而 LDHB 对乳酸有较高的亲和力, 并优先将乳酸转化为丙酮酸13。LDHA 启动子包含 HIF1α、cMYC 和 FOXM1 转录因子11的绑定站点。此外, 像许多其他酵酶14,15, LDH 可以修改后, 翻译修改。成纤维细胞生长因子受体1可以 phosphorylate LDHA 在 Y10, 促进聚丙烯形成, 或 Y83, 促进 NADH 基质结合16。LDHA 也可以乙酰17。由于这些原因, 对 ldh 活性的完全了解不仅需要监测 ldh 蛋白水平, 还要监控酶的活性。
除了我们在这里提出的方法外, 还使用了其他方法来监测乳酸脱氢酶的活性。乳酸脱氢酶活性可以监测 spectrophotometrically 在匀质蛋白裂解。NADH 乳酸转化为丙酮酸的世代可以根据 340 nm 的吸光度来测定, 而 NADH 的消失可以监测, 因为丙酮酸转化为乳酸18。乳酸脱氢酶活性也受到磁共振成像 (MRI) 监测。13丙酮酸盐可以被管理, 丙酮酸转化为乳酸可以被监测为 [1-13c] 乳酸/[1-13c] 丙酮酸的比例。1-13c] 乳酸/[1-13c] 丙酮酸盐的高比在癌症组织19被观察了。虽然 MRI 的方法可以提供关于正常和疾病组织中乳酸脱氢酶活性的信息, 但方法没有确定特定细胞活动水平所需的分辨率。这里提供的方法可以提供关于在组织区域甚至在单个细胞中乳酸脱氢酶活性的信息。
应用原位活性测定法, 发现在小鼠皮肤毛囊干细胞中乳酸脱氢酶活性高20。我们还利用该方法对培养的胚胎干细胞中乳酸脱氢酶的活性进行了监测, 发现干细胞的活性高于饲养层。最后, 对新鲜小鼠主动脉中乳酸脱氢酶的活性进行了监测, 观察了平滑肌细胞的染色。这里我们描述了一个详细的协议, 以监测冷冻小鼠皮肤乳酸脱氢酶活性。

<table border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:312px;">Surgical instruments</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;">For collecting skin from euthanized mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tissue-tek cryomold 25 mm x 20 mm x 5 mm</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">NC9511236</td>
			<td style="width:217px;">For freezing mouse skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">NC9638938</td>
			<td style="width:217px;">For mounting cryomolds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ice bucket</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">07-210-106</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Dry Ice</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Polysine Adhesion Slide</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">12-545-78</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">4% formalin</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">23-245-684</td>
			<td style="width:217px;">Dilluted in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">phosphate buffered saline, pH 7.4</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">vortex</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tris base</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">23-245-684</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NAD</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">N7004</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phenazine methosulfate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">P9625</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">Nitrotetrazolium blue chloride</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">N6876</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lithium L-lactate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">L2250</td>
			<td style="width:217px;">Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">37&#176;C incubator (or tissue culture incubator)</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Braziliant! Counter stain</td>
			<td style="width:164px;">Anatech</td>
			<td style="width:121px;">861</td>
			<td style="width:217px;">Counter stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mounting medium</td>
			<td style="width:164px;">Vector Laboratories</td>
			<td style="width:121px;">H-5000&#160;</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Cover slips for slides</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">12-544D</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light microscope</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
	</tbody>
</table>

mapping metabolism: monitoring lactate dehydrogenase activity directly in tissue

在空间和时间内绘制酶活性是理解正常组织和疾病细胞行为的分子基础的关键。原位代谢活性测定可以提供有关组织内代谢活动的空间分布的信息。我们提供了一个详细的协议, 以监测乳酸脱氢酶的活性直接在组织样品。乳酸脱氢酶是一种重要的决定因素, 即食用葡萄糖是否会通过有氧或厌氧糖酵解转化为能量。含有乳酸和 NAD 的溶液被提供给冰冻组织切片。乳酸脱氢酶活性高的细胞将提供的乳酸转化为丙酮酸, 同时将所提供的烟酰胺腺嘌呤二核苷酸 (NAD) 转化为 NADH 和质子, 可根据还原nitrotetrazolium 蓝色到 formazan, 这是可视化为蓝色沉淀。我们描述了一个详细的协议, 以监测乳酸脱氢酶活性的小鼠皮肤。应用该协议, 我们发现乳酸脱氢酶活性是高的静态毛囊干细胞在皮肤内。将该协议应用于培养的小鼠胚胎干细胞, 发现培养的胚胎干细胞比小鼠胚胎成纤维细胞有更高的染色。对刚分离的小鼠主动脉的分析显示, 垂直于主动脉的平滑肌细胞染色。所提供的方法可用于空间地映射在冷冻或新鲜组织中产生质子的酶活性。

我们以前报告的结果,在现场活性测定在老鼠皮肤20。如图 3所示, 我们观察到当上述程序被遵循时, 在毛囊底部毛囊干细胞中乳酸脱氢酶活性较高。这些发现通过荧光活化细胞分选皮肤的毛囊干细胞和确认高乳酸脱氢酶活性的干细胞室与活性检测的排序细胞。
根据我们的发现, 毛囊...

Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

映射代谢: 直接在组织中监测乳酸脱氢酶活性

我们描述了一种用于映射酶活性的空间分布的协议, 它能直接在组织样品中生成 nicotinatmide 腺嘌呤二核苷酸磷酸 (NAD (P) h) + h。

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Research

JoVE Journal

Biochemistry

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

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科研

生物化学

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用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Quantification of Hypopigmentation Activity In Vitro

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using ÃƒÂÃ¢â‚¬Â¡CRAC

使用χCRAC以高时间分辨率监测体内蛋白质-RNA相互作用动力学

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

大鼠胰腺组织快速、经济高效的RNA提取

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...