Cellular Creatine/Phosphocreatine Homeostasis

热力学的基本定律适用于生物体，就像它们适用于物理世界一样。因此，所有活着的细胞都必须代谢分解碳水化合物等大的有机分子，并在这个过程中捕获以一种名为三磷酸腺苷的化合物的形式释放的能量。三磷酸腺苷作为细胞的能量货币发挥作用。它的化学分解被用来驱动各种不利的能量过程，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，三磷酸腺苷供不应求，就像肌肉爆裂性收缩开始时一样。在许多细胞中，存在一种名为磷酸肌酸的化合物，它通过一种名为肌酸激酶的酶的作用，能够像使用ATP一样迅速地补充(至少在短期内)。磷酸肌酸的这种能量缓冲作用在广泛的细胞功能中是至关重要的。磷酸肌酸的前体是肌酸。在脊椎动物中，所需肌酸的一部分从饮食中获得，其余的在肝脏和胰腺中合成。然后，肌酸在血液中被运送到细胞，在那里它被一种特殊的膜转运蛋白(肌酸转运体)输送到细胞中，然后转化为磷酸肌酸。肌酸生物合成和运输的遗传缺陷会对人类产生严重的病理影响，包括严重的精神发育迟缓。许多不同的无脊椎动物群体在它们的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，例如海胆，它们是广泛使用的肥料。这些动物确实将卵子和精子排入海水中；这种策略需要产生大量的精子，这些精子含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，但完全不清楚这些动物是如何获得这种重要物质并将其输送到需要它的细胞的。拟议的研究工作试图通过研究选定的无脊椎动物群体中的这些过程来追踪肌酸生物合成和膜运输能力的演变。最初的努力将集中在1)使用敏感的同位素标记技术来确定肌酸生物合成的两个关键酶是否存在于这些动物中；以及2)使用现代分子遗传学方法来定位这些酶的组织表达，并确定这些蛋白质是否存在于生殖活动的高峰期(当对肌酸的需求很高时)。这项研究的第二个方面将集中在确定肌酸进入细胞的性质，在细胞中，肌酸以高浓度的肌酸/磷酸肌酸的形式积累。再一次，分子遗传学方法将被用来在细胞系统中表达运输蛋白，在那里它们可以被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械信息，并提供从低级无脊椎动物到更高级动物的这些过程的演变图景。

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.