Macromolecular Organization and Function of the Aqueous Cytoplasm

拟议中的研究是一项长期计划的继续 旨在了解其结构、性质和代谢 动物细胞水相区室的活性： 核质，细胞质膜结合细胞器的内部， 以及细胞质或含水细胞质。 虽然这些 车厢占大多数的质量和体积的75%左右 细胞，是代谢活动的场所，很少 我们知道他们的财产和组织的细节。 目前的工作重点是含水细胞质，并采用 主要培养的小鼠成纤维细胞（L细胞）， 用硫酸葡聚糖透化（DSP细胞）。 他们保留了大部分 大分子被释放到介质中， 处理（洗涤剂提取或均质化）：那些 通常认为可自由溶解的大分子 （“可溶的”）在水性细胞质中。 保留其中大部分 尽管有损伤（“孔”），但DSP细胞中的大分子仍然存在 在它们的质膜上，足够大， 逃跑 这项研究的目的是检验假设， 大多数“可溶性”大分子通过萃取和经典的 细胞分级分离在完整细胞中实际上不是游离的， 而是组织化，并与细胞基质松散地结合。 将在DSP细胞中通过以下检查来检验该假设： 糖酵解的酶（长期以来被认为是“可溶的”）使用一种 各种代谢探针和高压电子显微镜。 的 这些酶（和一般的蛋白质）从DSP流出 细胞暴露于旨在干扰假设的治疗 细胞基质酶协会将研究电泳 和酶活性测定。 DS透化的研究 也将启动其它细胞类型。 虽然我们对细胞质的组织结构知之甚少 活细胞在大分子水平上的作用， 作为一个严谨的研究课题被忽视，因为“细胞质” 通常被认为是蛋白质的简单溶液， 小分子。 最近的几条证据来自细胞 生物学和生物化学表明，这是一个过于简单化的问题 和误导性的观点。 拟议的研究将 为蛋白质的组织状态提供了急需的数据 以及细胞中的小分子， 不同的和更现实的概念细胞结构和 功能

The proposed research is the continuation of a long range program aimed at understanding the structure, properties and metabolic activities of the aqueous compartments of animal cells: nucleoplasm, interiors of cytoplasmic membrane-bound organelles, and the cytosol, or aqueous cytoplasm. Although these compartments make up about 75% of the mass and volume of most cells, and are the sites of much metabolic activity, very little is known about the details of their properties and organization. Current work focuses on the aqueous cytoplasm, and employs chiefly cultured mouse fibroblasts (L cells) which have been permeabilized with dextran sulfate (DSP cells). They retain most macromolecules that are released into the medium by harsher treatments (detergent extraction or homogenization): those macromolecules commonly thought to be freely dissolved ("soluble") in the aqueous cytoplasm. Retention of most of these macromolecules in DSP cells occurs in spite of lesions ("pores") in their plasma membranes that are large enough to allow them to escape. This research is designed to test the hypothesis that most "soluble" macromolecules defined by extraction and classical cell fractionation are not actually free in intact cells but are instead organized, and associated loosely with the cytomatrix. The hypothesis will be tested in DSP cells by an examination of the enzymes of glycolysis (long believed to be "soluble") using a variety of metabolic probes and high voltage electron microscopy. The efflux of these enzymes (and proteins in general) from DSP cells exposed to treatments designed to perturb postulated cytomatrix-enzyme associations will be studied by electrophoretic and enzyme activity assays. Studies on DS permeabilization of other cell types will also be initiated. Although little is known about the organization of the cytoplasm of living cells at the macromolecular level, it has long been overlooked as a subject of rigorous study because "cytoplasm" has generally been regarded as a simple solution of proteins and small molecules. Several lines of recent evidence from cell biology and biochemistry suggest that this is an oversimplified and misleading view of the cell. The proposed research will provide much needed data on the state of organization of proteins and small molecules in cells and could lead to a fundamentally different and more realistic concept of cellular structure and function.