MEMBRANES AND ORGANELLES-STRUCTURE AND FUNCTION

这项为期20年的计划旨在通过以下方面了解细胞功能 细胞器、膜和膜组件。重点放在……的层面 膜由两种纯化的膜组分D-β-羟基丁酸酯组成 脱氢酶是一种需要脂肪的酶，从心脏和 肝脏线粒体对卵磷脂有绝对需求 肌浆酶活性与钙泵蛋白(CPP) 快速抽动骨骼肌网，一种三磷酸腺苷驱动的膜泵 去除肌浆中的钙离子，使肌肉放松。这个 重建方法正在被用来制备定义的膜 成分、脂质环境和改变脂肪/蛋白质组成 膜。目前正在进行研究以阐明分子结构。 并将结构与功能的锻炼联系起来。我们 继续指导各种方法，包括生化和 生物物理学，以表征脂-蛋白质相互作用的性质 膜包括：1)尝试获得二维和三维 膜蛋白的结晶使这些成分的结构 2)脂类环境影响的研究。 功能；3)用氢气、质子和磷核磁共振测量运动 磷脂的特性与“界”的汇率 具有双层磷脂的磷脂。对于BDH来说，一个关键问题是 脂类作用的基础；这种脱氢酶区别于 许多其他的功能不需要类脂。化学制品 衍生研究和初级序列分析正在进行中。 还研究了含双氢呋喃的取向多层膜的衍射谱 设计用来揭示BDH在膜中的取向。我们之前 发现在糖尿病患者中，精选脂肪酸的饱和度增加 肝脏线粒体膜发生酯化为磷脂 在胰岛素剥夺后一周内，BDH水平下降超过 几个星期的时间。这一下降的基础将被研究。一个 第二个主要目的是在快速抽动的骨骼肌中定义 兴奋-收缩耦合中钙离子释放过程的本质。这个 方法是分离和研究高纯度的膜组分 包括质膜、顶池和纵池、横池 小管和三联体(终池和三联体的连接 横管)。重点是用分子术语来定义 三联体结合的性质和钙离子释放的方式 通过这个细胞内连接被触发。

This program of twenty years aims to understand cell function in terms of organelles, membranes and membrane components. The focus on the level of the membrane is on two purified membrane components, D-Beta-hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, prepared from heart and liver mitochondria which has an absolute requirement for lecithin for enzymic activity and the calcium pump protein (CPP) from sarcoplasmic reticulum of fast-twitch skeletal muscle, an ATP-driven membrane pump which removes Ca2+ from the sarcoplasm enabling muscle to relax. The reconstitution approach is being used to prepare membranes of defined components, lipid environment and to vary the lipid/protein composition of the membrane. Studies are ongoing to elucidate the molecular architecture of the membrane and to relate structure to the exercise of function. We continue directing a variety of approaches, both biochemical and biophysical, to characterize the nature of lipid-protein interactions in membranes including: 1) attempts to obtain two- and three-dimensional crystals of the membrane proteins so that the structure of these components can be elucidated; 2) studies of the influence of the lipid environment on function; 3) deuterium, proton and phosphorus NMR to measure the motional characteristics of the phospholipid and the exchange rate of "boundary" phospholipid with bilayer phospholipid. For BDH, a key question is the basis of the role of lipid; what makes this dehydrogenase distinct from many others which do not require lipid for function. Chemical derivatization studies and primary sequence analysis are in progress. Diffraction studies of oriented multilayers containing BDH are also designed to reveal orientation of BDH in the membrane. We previously found, in diabetes, increases in the saturation of select fatty acids esterified to phospholipid in the mitochondrial membrane of liver occurred within a week after insulin deprivation and the level of BDH decreased over a period of several weeks. The basis for this decrease will be studied. A second major thrust is to define, in fast-twitch skeletal muscle, the nature of the Ca2+ release process in excitation-contraction coupling. the approach is to isolate and study highly purified membrane components including plasmalemma, terminal and longitudinal cisternae, transverse tubule, and triads (the junctional association of terminal cisternae and transverse tubule). The emphasis is to define, in molecular terms, the nature of the triad junctional association and how Ca2+ release is triggered across this intracellular junction.