Bioluminescence:Molecular Mechanisms and Biochemical Control

这项研究将侧重于海洋甲藻（负责“海洋磷光”）和陆地双翅目（苍蝇）发光系统的基本生化机制，这是两个主要的和进化上独立的生物发光生物群体。甲藻Gonyaulax polyedra的发光反应的生化成分-荧光素酶（LCF），荧光素底物（LH 2），独特的四吡咯和荧光素结合蛋白（LBP）-被隔离在一个细胞器，荧光素酶。LCF由三个高度保守和连续的序列组成，每个序列都具有催化活性。将确定活性所需的最小肽大小和活性位点的位置。比较LCF序列从其他生物发光甲藻将允许残基保守的酶活性和调节被确定，我们的理解的荧光素酶基因结构的演变将增加。北美双翅目双翅目扁尾虫（Platyura fultoni，长15毫米）是结网的幼虫，有两个发光器官（头部和尾部），并发出蓝色的荧光，发射峰位于460 nm，但生物发光反应的生物化学尚不清楚。氧气是必需的，这表明在所有生物发光反应中，过氧化物中间体的参与。据报道，位于这些器官中的巨细胞的线粒体的颗粒分泌与光发射有关。这项研究将侧重于这些不寻常的细胞学方面和生物化学成分的鉴定。织网幼虫的澳大利亚生物发光双翅目，Arachnocampa黄，目前的相似性与Platyura尽管光器官解剖的差异，这两个双翅目的发光反应的生物化学进行比较。了解生物发光的基本机制将促进能量代谢的知识，以及我们对涉及活性氧的过程的理解，现在已知活性氧作为细胞信使以及细胞损伤的试剂。 对生物发光的基因、酶和底物的基础知识已经导致了广泛的应用，从基因表达的灵敏可视化到药物和代谢物的分析测量。 甲藻的研究，这里提出的承诺，在酶学，以及在分子进化的重大进展。 研究人员最近开始研究的双翅目生物发光的生物化学是完全新颖的，因此充满了新知识和应用的希望。 该项目的具体目标如下：（a）检查其他生物发光甲藻的荧光素酶基因，以便深入了解基因进化，并帮助确定参与催化的氨基酸残基;（B）确定荧光素酶活性在各个结构域内所需的结构区域;（c）表征双翅目扁尾两栖类和蛛形纲的生物发光反应的生化组分，分离并部分纯化荧光素酶和荧光素酶;测试甲虫荧光素酶与双翅目昆虫荧光素反应的能力。

This research will focus on the fundamental biochemical mechanisms of light emitting systems in marine dinoflagellates (responsible for the "phosphorescence of the sea") and terrestrial Diptera (flies), two major and evolutionarily independent groups of bioluminescent organisms. The biochemical components of the light-emitting reaction of the dinoflagellate Gonyaulax polyedra - the luciferase enzyme (LCF), the luciferin substrate (LH2), a unique tetrapyrrole, and a luciferin binding protein (LBP)- are sequestered in an organelle, the scintillon. LCF consists of three highly conserved and contiguous sequences, each of which is catalytically active. The minimum peptide size necessary for activity and the location of the active site will be determined. Comparison of LCF sequences from other bioluminescent dinoflagellates will allow residues conserved for enzyme activity and regulation to be identified, and our understanding of the evolution of the luciferase gene structure will be increased. Larvae of the web-building, carnivorous North American dipteran Platyura fultoni (15 mm long) have two light organs (head and tail) and emit a blue luminescence with an emission peak at 460 nm, but the biochemistry of the bioluminescence reaction is not known. Oxygen is required, suggesting the involvement of a peroxidic intermediate, as in all bioluminescence reactions. A granular secretion ascribed to the mitochondria of giant cells located in these organs is reported to be associated with the light emission. The study will focus on these unusual cytological aspects and on the identification of the biochemical components. The web-weaving larvae of an Australian bioluminescent dipteran, Arachnocampa flava, present similarities with Platyura despite differences in light organ anatomy; the biochemistry of the light emitting reactions of these two diptera will be compared. An understanding of the basic mechanisms in bioluminescence will advance the knowledge of energy metabolism, as well as our understanding of processes involving reactive oxygen species, now known to serve as cellular messengers as well as agents of cellular damage. Fundamental knowledge of the genes, enzymes and substrates responsible for bioluminescence has led to extensive applications, ranging from sensitive visualization of gene expression to analytical measurements of drugs and metabolites. The dinoflagellate studies proposed here promise to contribute significant advances in enzymology, as well as in molecular evolution. The biochemistry of the dipteran bioluminescence, which the investigator recently began studying, is entirely novel, hence rich in promises of new knowledge and applications. Specific aims for this project are as follows: (a) examine luciferase genes from other bioluminescent dinoflagellates so as to gain insight into gene evolution and to aid in the determination of amino acid residues involved in catalysis; (b) define the structural regions required for luciferase activity within the individual domains; (c) characterize the biochemical components of the bioluminescence reactions of the dipterans Platyura and Arachnocampa, isolate and partially purify luciferin and luciferase; test for the ability of the beetle luciferase to react with the dipteran luciferin.