Cellular Creatine/Phosphocreatine Homeostasis

热力学的基本定律既适用于物质世界，也适用于有机体。因此，所有活细胞都必须代谢分解碳水化合物等大型有机分子，并在此过程中捕获以三磷酸腺苷（简称ATP）形式释放的一些能量。ATP是细胞的能量货币。它的化学分解被用来驱动各种能量不利的过程，如生物合成，细胞运动，如收缩，以及将物质向上泵过生物膜。有些情况下，对ATP的需求超过供应，这可能发生在爆发性肌肉收缩的开始。在许多细胞中存在一种称为磷酸肌酸的化合物，它通过一种称为肌酸激酶的酶的作用，能够像ATP被使用一样迅速地补充ATP（至少在短期内）。这种能量缓冲作用的磷酸肌酸是至关重要的功能，广泛的细胞。磷酸肌酸的前体是肌酸。在脊椎动物中，所需肌酸的一小部分从饮食中获得，其余部分在肝脏和胰腺中合成。肌酸随后在血液中被运送到细胞中，在细胞中由一种特殊的膜转运蛋白（肌酸转运蛋白）运送到细胞中，然后转化为磷酸肌酸。肌酸生物合成和运输的遗传缺陷在人类中产生严重的病理影响，包括严重的智力迟钝。许多不同的无脊椎动物群体在细胞中积累了大量的肌酸/磷酸肌酸。对于某些海洋物种来说尤其如此，比如海胆，它们是一种“广播肥料”。这些动物真的把它们的卵子和精子丢进海水里；这种策略需要产生大量的精子，这些精子具有非常高水平的肌酸/磷酸肌酸。因此，对肌酸的季节性需求很高，但这些动物是如何获得并将这种重要物质运送到需要它的细胞的，目前还不清楚。拟议的研究努力旨在通过在选定的无脊椎动物群体中调查这些过程来追踪肌酸生物合成和膜运输能力的进化。最初的工作将集中在1)使用敏感的同位素标记技术来确定这些动物体内是否存在肌酸生物合成的两种关键酶；2)使用现代分子遗传学方法定位这些酶的组织表达，并确定在生殖活动高峰期（当对肌酸的需求很高时）这些蛋白质的存在是否增加。这项研究的第二个方面将集中于确定肌酸运输到细胞中的性质，在细胞中它以高浓度的肌酸/磷酸肌酸积累。再一次，分子遗传学方法将用于表达运输蛋白在细胞系统中，他们可以最有效地研究和表征。总体结果将提供有关肌酸生物合成和运输的机制信息，并提供这些过程从低级无脊椎动物到更高级动物的进化图景。

The basic laws of thermodynamics apply to organisms as they do to the physical world. As a consequence, all living cells must metabolically breakdown large organic molecules such as carbohydrates and in the process trap some of the energy released in the form of a compound known as adenosine triphosphate, ATP for short. ATP functions as the cell's energy currency. Its chemical breakdown is used to drive a variety of energetically unfavorable processes such as biosynthesis, cellular movement like contraction, and pumping substances uphill across biological membranes. There are instances where demand for ATP exceeds supply as might take place at the onset of burst muscle contraction. In many cells a compound known as phosphocreatine is present which, through the action of an enzyme called creatine kinase, is capable of replenishing ATP as quickly as it is used (at least in the short term). This energy buffering role of phosphocreatine is critical in the functioning of a broad spectrum of cells. The precursor for phosphocreatine is creatine. In vertebrates, a fraction of the creatine required is obtained from the diet while the rest is synthesized in the liver and pancreas. Creatine is then carried in the blood to cells where it is transported by a special membrane transport protein (creatine transporter) into the cell and then converted to phosphocreatine. Genetic defects in creatine biosynthesis and transport produce severe pathological effects in humans including profound mental retardation. Many diverse invertebrate groups accumulate large quantities of creatine/phosphocreatine in their cells. This is particularly true of certain marine species such as sea urchins which are broad-cast fertilizers. These animals literally shed their eggs and sperm into to the sea water; this strategy necessitates production of massive amounts of sperm which have very high levels of creatine/phosphocreatine. Thus, there is a seasonally high demand for creatine yet it is not clear at all how these animals obtain and transport this vital substance to cells where it is needed. The proposed research effort seeks to trace the evolution of creatine biosynthetic and membrane transport capacities by investigating these processes in selected groups of invertebrates. Initial efforts will focus on 1) using sensitive isotopic labeling techniques to determine whether the two key enzymes of creatine biosynthesis are present in these animals; and 2) using modern molecular genetic approaches to localize the tissue expression of these enzymes, and to determine whether there is increased presence of these proteins at peak reproductive activity (when demand for creatine is high). A second facet of this research will center on determining the nature of creatine transport into cells where it accumulates as high concentrations of creatine/phosphocreatine. Once again, molecular genetic methods will be used to express the transport proteins in a cell system where they can be studied and characterized most efficiently. The overall results will yield mechanistic information about creatine biosynthesis and transport as well as provide a picture of the evolution of these processes from the lower invertebrates to more advanced animals.