MEMBRANES AND ORGANELLES-STRUCTURE AND FUNCTION

这项为期20年的计划旨在了解细胞功能， 细胞器、膜和膜组分。 重点放在 该膜是在两个纯化的膜组件，D-β-羟基丁酸 脱氢酶（BDH）是一种需要脂质的酶，从心脏和 肝线粒体对卵磷脂有绝对的需求， 酶活性和肌浆钙泵蛋白（CPP） 快速收缩骨骼肌的网状结构，一种ATP驱动的膜泵， 去除肌浆中的Ca 2+，使肌肉放松。 的 重构方法被用于制备限定的膜， 成分，脂质环境，并改变脂质/蛋白质组成， 膜。 研究正在进行中，以阐明分子结构 并将结构与功能的行使联系起来。 我们 继续指导各种方法，包括生物化学和 生物物理，以表征脂质-蛋白质相互作用的性质， 膜，包括：1）试图获得二维和三维 膜蛋白的晶体， 2）研究脂质环境对 3）氘、质子和磷NMR来测量分子的运动 磷脂的特性与交换率的“边界” 磷脂与双层磷脂。 对于BDH来说，一个关键问题是 脂质作用的基础;是什么使这种脱氢酶不同于 许多其他的不需要脂质来发挥功能。 化学 衍生化研究和初级序列分析正在进行中。 含BDH取向多层膜的衍射研究也 旨在揭示BDH在膜中的取向。 我们之前 在糖尿病患者中， 肝线粒体膜磷脂发生酯化反应， 在胰岛素剥夺后一周内，BDH水平下降超过 几周的时间。 将研究这一减少的原因。 一 第二个主要目标是在快速收缩的骨骼肌中， 兴奋-收缩偶联中Ca 2+释放过程的性质。 的 方法是分离和研究高度纯化的膜组分 包括质膜，顶池和纵池，横池， 小管和三联体（终末池和 横小管）。 重点是在分子方面定义， 三联体连接缔合的性质以及Ca 2+释放是如何 在细胞内连接处触发。

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.