Novel enzymatic activities of the bioluminescent protein, luciferase

生物发光蛋白荧光素酶的新型酶活性

• 批准号: 8736506
• 负责人: Steven Sollott
• 金额: $ 8.61万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736506
• 关键词: American; Animals; Bacteria; Behavior; Biochemical; Biological; Biological Assay; Bioluminescence; Chemiluminescence assay; Column Chromatography; Communication; Complex; Cytoplasm; Detection; Diphosphates; Environment; Enzymes; Event; Fireflies; Gel; Generations; Image; Imaging Techniques; Immunoblotting; Incubated; Ions; Kinetics; Light; Luciferases; Luminescent Proteins; Marines; Measurement; Molecular Probes; Morphologic artifacts; Muscle; Nucleotides; Output; Oxygen; Peptidylprolyl Isomerase; Photinus; Photons; Plants; Preparation; Production; Proteins; Proteomics; Reaction; Recombinant Proteins; Recombinants; Reporting; Research; Running; Signal Transduction; Specificity; Staining method; Stains; System; Testing; Uncertainty; adenylate kinase; design; inorganic phosphate; luciferin; novel; quantum; single molecule; tool

Luciferases are a class of proteins expressed in a wide variety of terrestrial and marine animal and plant species that enable communication of signals via bioluminescence. One of the most familiar such systems is known from the American firefly, Photinus pyralis. The enzymatic activity of luciferase is activated by ATP and catalyzes the reaction of oxygen and the substrate, luciferin, releasing energy as a visible photon together with the byproducts, AMP, pyrophosphate and oxidized luciferin. Chemiluminescence is one of the most sensitive ways to probe molecular mechanisms because, (1) the conversion of the biochemical energy of ATP into light by this enzyme has a quantum yield of approximately 40%, and (2) single photon counting can be performed using advanced low-light-level detection systems, enabling the recording and study of events from single molecule reactions. As noted above, there are unexplained complexities in the behavior of luciferase, and in particular, we have noted certain paradoxical levels of light production and non-linearity occur when the enzyme is exposed to its known substrate, ATP, in the presence of other nucleotides, such as ADP. Indeed, it has been reasoned that much of this mechanistic uncertainty could be explained by a certain level of luciferase contamination with adenylate kinase (a.k.a., "myokinase"), which is abundant in preparations from which luciferase is purified (e.g., contaminated by the flight muscles in fireflies, as well as from the cytoplasm of bacteria used in recombinant protein generation, etc.). Adenylate kinase produces ATP through the dismutation of ADP, which would produce an unforseen and uncontrolled contaminating light signal in the luciferase reaction independent of the ATP originally present and thus confounding the accuracy and specificity of the measurement. We sought to examine the catalytic activity and chemiluminescence mechanisms of luciferase in the presence of ADP. We found that under certain circumstances where ADP is in quantitative excess over ATP, there can be a significant excess light output vs that expected quantitatively from pure ATP standards. To rule out that adenylate kinase contamination was responsible for this apparent artifact, we purified several luciferases (both from firefly extracts and recombinant material) using preparative column chromatography designed to exclude < 30 kD species (adenylate kinase mw approx. 20 kD). After confirming the quantitative depletion of species < 30 kD, we found that the ADP-related artifact on luciferase light output was still present, indicating that adenylate kinase contamination was not the cause. It has been reported elsewhere that inorganic pyrophosphate (PPi) may increase the output of luciferase bioluminescence. To rule out that the possible activating effect of ADP in our bioluminescence assay was an artifact caused by the presence of PPi contamination in the preparations used, we treated the ADP with inorganic pyrophospatase (PPiase) that converts one molecule of pyrophosphate to two phosphate ions. PPiase treatment although changing the kinetics of the light production had no effect on the total light output. Furthermore, we demonstrated that addition of external PPi to ADP did not activate but instead significantly inhibited luciferase light production and this inhibition was reversed by treatment with PPiase. Thus, we ruled out an artifact produced by PPi contamination. We then hypothesized that luciferase itself might be able to obtain sufficient energy from ADP alone to produce the light-output artifact. In order to test this, we developed an in-gel chemiluminescence assay run on a clear native gel platform. The luciferase was first separated from other potential enzyme contaminants using a clear native gel. This gel was subsequently incubated with purified ADP and luciferin, and low light level imaging showed that 2 discrete bands yielded significant light output. Subsequent addition of an excess of ATP to this mixture showed that these exact same bands that produced light with ADP alone, were also producing substantially increased light with the (excess) ATP. At the end of the imaging procedure, immunoblotting of this gel confirmed that the bands producing light were positively stained for luciferase (and negative for adenylate kinase), and proteomic analysis of these bands (from gel lanes not used for immunoblotting) identified that the only protein present was luciferase without any other protein contamination. We conclude that luciferase itself is capable of using the energy of ADP to produce light output independently of the presence of preformed ATP, possibly by the catalytic conversion of ADP to ATP and the latter being utilized as the ultimate substrate.

荧光素酶是一类在多种陆地和海洋动物和植物物种中表达的蛋白质，能够通过生物发光进行信号通信。 最熟悉的此类系统之一是美国萤火虫 Photinuspyralis。 荧光素酶的酶活性由 ATP 激活，并催化氧和底物荧光素的反应，以可见光子的形式释放能量以及副产物 AMP、焦磷酸盐和氧化荧光素。 化学发光是探测分子机制最灵敏的方法之一，因为 (1) 这种酶将 ATP 生化能转化为光的量子产率约为 40%，(2) 单光子计数可以使用先进的微光检测系统，能够记录和研究单分子反应的事件。如上所述，荧光素酶的行为存在无法解释的复杂性，特别是，我们注意到，当酶在其他核苷酸存在的情况下暴露于其已知底物 ATP 时，会出现某些自相矛盾的光产生水平和非线性。 ，例如ADP。 事实上，据推测，这种机制上的不确定性很大程度上可以通过一定程度的腺苷酸激酶（又名“肌激酶”）的荧光素酶污染来解释，腺苷酸激酶（又名“肌激酶”）在纯化荧光素酶的制剂中含量丰富（例如，被飞行污染）萤火虫的肌肉，以及用于重组蛋白生成的细菌的细胞质等）。 腺苷酸激酶通过 ADP 歧化产生 ATP，这会在荧光素酶反应中产生不可预见且不受控制的污染光信号，与最初存在的 ATP 无关，从而影响测量的准确性和特异性。 我们试图研究 ADP 存在下荧光素酶的催化活性和化学发光机制。 我们发现，在 ADP 定量超过 ATP 的某些情况下，与纯 ATP 标准品的预期定量相比，可能会出现显着过量的光输出。 为了排除腺苷酸激酶污染导致这种明显的伪影，我们使用旨在排除 < 30 kD 物种（腺苷酸激酶分子量约为 20 kD）的制备柱色谱纯化了几种荧光素酶（来自萤火虫提取物和重组材料）。 在确认 < 30 kD 的物种定量耗尽后，我们发现荧光素酶光输出上与 ADP 相关的伪影仍然存在，表明腺苷酸激酶污染不是原因。据其他地方报道，无机焦磷酸盐 (PPi) 可能会增加荧光素酶生物发光的输出。为了排除我们的生物发光测定中 ADP 可能的激活作用是由所用制剂中存在 PPi 污染引起的人为因素，我们用无机焦磷酸酶 (PPiase) 处理 ADP，该酶将一个焦磷酸分子转化为两个磷酸根离子。 PPiase 处理虽然改变了光产生的动力学，但对总光输出没有影响。此外，我们证明，在 ADP 中添加外部 PPi 不会激活荧光素酶光的产生，而是会显着抑制荧光素酶光的产生，并且通过 PPiase 处理可以逆转这种抑制。 因此，我们排除了 PPi 污染产生的伪影。 然后我们假设荧光素酶本身可能能够单独从 ADP 获得足够的能量来产生光输出伪影。 为了测试这一点，我们开发了一种在透明的天然凝胶平台上运行的凝胶内化学发光测定法。 首先使用透明的天然凝胶将荧光素酶与其他潜在的酶污染物分离。 随后将该凝胶与纯化的 ADP 和荧光素一起孵育，低光水平成像显示 2 个离散条带产生显着的光输出。 随后向该混合物中添加过量的 ATP 表明，这些与单独使用 ADP 产生光的完全相同的条带，也使用（过量）ATP 产生显着增加的光。 在成像过程结束时，对该凝胶的免疫印迹证实，产生光的条带对荧光素酶染色呈阳性（对腺苷酸激酶染色呈阴性），并且对这些条带（来自未用于免疫印迹的凝胶泳道）进行蛋白质组学分析，确定唯一的存在的蛋白质是荧光素酶，没有任何其他蛋白质污染。 我们得出的结论是，荧光素酶本身能够利用 ADP 的能量产生光输出，而与预先形成的 ATP 的存在无关，可能是通过 ADP 催化转化为 ATP 并将后者用作最终底物。