Development of a Molecular Probe for Investigating Cell- Surface Enzyme Activity in Seawater

氮是浮游植物生长所必需的营养物质之一。氮素循环的速度可以决定初级生产力的速度。海洋氮素循环的主要途径之一是浮游植物吸收氮，并将其并入生命所需的氨基酸和核酸中。传统上，海洋科学家认为浮游植物从无机氮化合物中获得所需的氮。然而，与无机氮化合物相比，溶解的有机氮化合物构成了海水中更大的氮库。此前，实地研究表明，浮游植物不会在很大程度上吸收氨基酸和其他有机氮化合物。最近有人提出，一些浮游植物可以利用细胞表面的一种酶来氧化胺和氨基酸。氨基酸的氧化产生过氧化氢、一种x-酮酸和铵。然后，氨离子通过浮游植物细胞膜运输，用作氮源。X-酮酸被释放到周围的海水中，在那里它可以被细菌吸收。如果通过机制氧化氨基酸的能力在浮游植物中普遍存在，这将对我们理解海水中氨基酸的循环以及浮游植物和细菌对这些化合物的竞争具有深远的意义。该项目将开发一系列荧光分子探针来模拟海水中的氨基酸和胺。利用高效液相色谱结合荧光检测，可以快速、灵敏地监测海水中探针的损失(由藻类氧化引起)。利用这些合成的探针，将在野外测试浮游植物通过这种新的机制氧化胺和氨基酸的重要性。

Nitrogen is one of the nutrients essential for phytoplankton growth. The rate of nitrogen recycling can determine the rate of primary production. One of the major pathways of nitrogen cycling in the sea is the uptake of N by phytoplankton for incorporation into the amino acids and nucleic acids required for life. Traditionally, marine scientists have thought that phytoplankton obtain the N they require from inorganic nitrogen compounds. However, dissolved organic nitrogen compounds make up a larger reservoir of N in seawater than do inorganic nitrogen compounds. Previously, field studies have indicated that amino acids and other organic nitrogen compounds are not taken up to any significant extent by phytoplankton. It has been recently suggested that some phytoplankton can oxidize amines and amino acids using a cell-surface enzyme. Oxidation of amino acids produces hydrogen peroxide, an x-keto acid, and ammonium. The ammononium is then transported across the phytoplankton cell membrane for use as a N source. The x- keto acid is released to the surrounding seawater where it can be taken up by bacteria. If the ability to oxidize amino acids by mechanism is widespread among phytoplankton, it has profound implications for our understanding of the cycling of amino acids in seawater and the competition between phytoplankton and bacteria for these compounds. This project will develop a series of fluorescent molecular probes to simulate amino acids and amines in seawater. Loss of the probes (by algal oxidation) from seawater can be monitored quickly and sensitively by high performance liquid chromatography with fluorescent detection. Using these synthesized probes, the importance of phytoplankton oxidation of amines and amino acids by this novel mechanism will be tested in the field.