RUI: Sphingolipid Metabolism in Plants

先前对植物鞘脂代谢中酶促反应的表征为本研究奠定了基础。参与鞘脂合成和转化的单个酶促步骤的细胞内位置将通过细胞分离和标记测定在豆类和玉米组织中确定。还将确定分离膜组分中鞘脂的具体含量和组成。标记鞘脂通过细胞的转移将通过鞘脂的体内标记和分离程序进行耦合跟踪。使用这种方法，将获得关于细胞内鞘脂运输的途径和动力学的信息。通过检测反应对蛋白水解酶的敏感性，研究完整膜泡中糖基神经酰胺合成的拓扑结构。此外，steryl gluco苷作为该反应的葡萄糖供体的作用将进一步研究。最后，催化合成神经酰胺磷酸化肌醇的酶促反应，肌醇磷酸化神经酰胺的生物合成前体，将在大豆和玉米的微粒体制备中得到证明和表征。随后，将对其亚细胞分布和拓扑结构进行表征。提出的研究将提供植物鞘脂代谢、细胞内运输/区隔以及被认为参与建立糖基神经酰胺在质膜中的不对称跨双层分布的关键酶的拓扑结构的综合观点。它还将提供关于合成肌醇磷酸化神经酰胺的第一步的新信息，肌醇磷酸化神经酰胺是植物中一类复杂的鞘脂。鞘脂存在于除细菌外的所有生物中，并作为细胞结构成分和各种生物过程的调节剂发挥作用。然而，我们对鞘脂在植物中的功能和代谢知之甚少。这项被提议的研究是由美国国家科学基金会支持的先前工作的延续，将提供鞘脂在细胞中产生、运输和降解的位置和方式的综合观点。它还将首次研究肌醇磷酸化神经酰胺的合成，这是一种新型和多样化的复杂植物脂类。这些研究的结果将有助于理解这些不寻常的脂质在植物生长发育中的作用。

9723484 Lynch The previous characterization of enzymatic reactions involved in sphingolipid metabolism in plants serves as a foundation for the proposed research. The intracellular locations of the individual enzymatic steps involved in sphingolipid synthesis and turnover will be determined in bean and corn tissues using cell fractionation and marker assays. The specific sphingolipid content and composition in the isolated membrane fractions will also be determined. The transfer of labeled sphingolipids through the cell will be tracked by coupling in vivo labeling of sphingolipids and fractionation procedures. Using this approach, information on the route and kinetics of intracellular sphingolipid transport will be obtained. The topology of glucosylceramide synthesis in intact membrane vesicles will be investigated by examining the sensitivity of the reaction to proteolytic enzymes. As well, the role of steryl glucoside as the glucose donor for this reaction will be further investigated. Finally, the enzymatic reaction catalyzing the synthesis of ceramide phosphorylinositol, the biosynthetic precursor for inositol phosphorylceramides, will be demonstrated and characterized in microsomal preparations from bean and corn. Subsequently, its subcellular distribution and topology will be characterized. The proposed research will provide an integrated view of plant sphingolipid metabolism, intracellular transport/compartmentation, and the topology of key enzymes thought to be involved in establishing the asymmetric transbilayer distribution of glucosylceramide in the plasma membrane. It will also provide new information about the first step in the synthesis of inositol phosphorylceramides, a diverse class of complex sphingolipids in plants. Sphingolipids are present in all organisms except bacteria and are known to function as structural components in cells and as regulators of various biological processes. Little is known, however, about the function and metabolism of sphingolipids in plants. The proposed research, a continuation of previous work supported by the NSF, will provide an integrated view of where and how sphingolipids are produced, transported, and degraded in the cell. It will also investigate, for the first time, the synthesis of inositol phosphorylceramides, a novel and diverse class of complex plant lipids. The results from these studies will contribute to understanding the role of these unusual lipids in plant growth and development.