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: 7963900
• 负责人: Steven Sollott
• 金额: $ 9.04万
• 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/7963900
• 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; cofactor; 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 catalyzes the reaction of ATP, oxygen and the cofactor, 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 90%, 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，其能够通过生物发光进行信号通信。 其中最熟悉的系统是从美国萤火虫，Photinus pyralis。 荧光素酶的酶活性催化ATP、氧和辅因子（辅蛋白）的反应，以可见光子的形式释放能量，同时释放副产物AMP、焦磷酸盐和氧化的辅蛋白。 化学发光是探测分子机制的最灵敏的方法之一，因为（1）ATP的生化能量通过这种酶转化为光的量子产率约为90%，以及（2）单光子计数可以使用先进的低光水平检测系统进行，从而能够记录和研究单分子反应的事件。如上所述，在荧光素酶的行为中存在无法解释的复杂性，并且特别地，我们已经注意到当酶在其他核苷酸（例如ADP）存在下暴露于其已知底物ATP时，发生某些矛盾水平的光产生和非线性。 事实上，已经推断，这种机制的不确定性中的大部分可以通过一定水平的荧光素酶与腺苷酸激酶（a.k.a.，“肌激酶”），其在从中纯化荧光素酶的制剂中是丰富的（例如，被萤火虫中的飞行肌肉污染，以及来自用于重组蛋白产生的细菌的细胞质等）。 腺苷酸激酶通过ADP的歧化产生ATP，这将在荧光素酶反应中产生不可预见和不受控制的污染光信号，与最初存在的ATP无关，从而混淆测量的准确性和特异性。 我们试图研究的催化活性和荧光素酶在ADP的存在下的化学发光机制。 我们发现，在某些情况下，ADP是在定量超过ATP，可以有一个显着的过量光输出与预期定量从纯ATP标准。 为了排除腺苷酸激酶污染是造成这种明显假象的原因，我们使用设计为排除< 30 kD物质（腺苷酸激酶分子量约为100 kD）的制备柱色谱法纯化了几种腺苷酸酶（来自萤火虫提取物和重组材料）。20 kD）。 在确认< 30 kD物质的定量消耗后，我们发现荧光素酶光输出上的ADP相关伪影仍然存在，表明腺苷酸激酶污染不是原因。据报道，无机焦磷酸盐（PPi）可以增加荧光素酶生物发光的输出。为了排除ADP在我们的生物发光测定中可能的活化作用是由所用制剂中PPi污染的存在引起的伪影，我们用无机焦磷酸酶（PPiase）处理ADP，该酶将一个焦磷酸盐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，加入外部PPi ADP没有激活，而是显着抑制荧光素酶的光产生，这种抑制被逆转的PPiase处理。 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，我们开发了在透明天然凝胶平台上运行的凝胶内化学发光测定。 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和白蛋白一起孵育，并且低光水平成像显示2个离散条带产生显著的光输出。 随后向该混合物中加入过量的ATP表明，这些与单独使用ADP产生光的完全相同的条带也在（过量）ATP的情况下产生显著增加的光。 在成像程序结束时，该凝胶的免疫印迹证实，产生光的条带对于荧光素酶是阳性染色的（而对于腺苷酸激酶是阴性的），并且这些条带的蛋白质组学分析（来自未用于免疫印迹的凝胶泳道）鉴定出存在的唯一蛋白质是荧光素酶，而没有任何其他蛋白质污染。 我们的结论是，荧光素酶本身能够使用ADP的能量产生光输出的存在下，预先形成的ATP独立，可能是由ADP的催化转化为ATP和后者被用作最终的底物。