ENZYME REGULATION AT FERTILIZATION OF SEA URCHIN EGGS

海胆卵受精时的酶调节

• 批准号: 3330717
• 负责人: DAVID EPEL
• 金额: $ 11.7万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3330717
• 关键词: affinity chromatography; allosteric site; aminoacid metabolism; antibody; binding proteins; bioenergetics; clathrate; crosslink; cytology; early embryonic stage; egg /ovum; embryo /fetus protein; embryogenesis; enzyme activity; fertilization; glucose 6 phosphate dehydrogenase; immunocytochemistry; invertebrate embryology; protein purification; protein sequence; protein structure; sea urchins; western blottings

The long term goal of this work is to understand regulation of enzyme activity upon fertilization, using sea urchin oocytes as a model system for studying activation. The proposed research will investigate an apparent activation of enzymes at fertilization, particularly glucose-6-phosphate dehydrogenase (G6PDH). This enzyme change is not seen in cell homogenates but is seen in vitro as a binding change, in permeabilized cells as an activity change and in vivo as an activity change. Comprehension of this phenomena will lead to greater understanding of egg activation at fertilization in particular and could be applicable to other cases of signal transduction, e.g., the action of growth factors on cells, in general. The objectives are to (1) identify the structural elements associated with G6PDH using crosslinking reagents or affinity chromatography, (2) characterize these proteins and their cDNAs, (3) use partial amino acid sequences to prepare antibodies and study the cytological locale of these proteins and any changes in locale and (4) also use the antibodies to examine any chemical modifications as assessed with Western blots. The research will also ascertain (5) whether the dissociation might be affected by small molecules produced or destroyed at fertilization and as assessed by enriching the permeabilization medium for endogenous small molecules. An ancillary but related goal is to extend new technologies for measuring enzyme activity in vivo by using caged substrates to ascertain (6) whether amino acid catabolism is an important energy source for development. All oocytes, even those of mammals, have yolk but the role of this yolk in supporting early development is not yet appreciated. This work with caged substrates will extend this technology and also provide new insights into the energetics of development.

这项工作的长期目标是了解酶的调节 活性受精，使用海胆卵母细胞作为模型系统 来研究激活。 这项研究将调查一个 受精时酶的表观活化，特别是 葡萄糖-6-磷酸脱氢酶（G6 PDH）。 这种酶的变化不是 在细胞匀浆中观察到，但在体外观察到结合变化， 透化细胞作为活性变化和体内作为活性 变化 理解这种现象将导致更大的 特别是对受精时卵子激活的理解， 可应用于信号转导的其它情况，例如，的作用 细胞生长因子的作用。 目标是：（1）确定相关的结构要素 使用交联试剂或亲和色谱法与G6 PDH，（2） 对这些蛋白及其cDNA进行了鉴定，（3）用部分氨基酸 序列来制备抗体并研究这些抗体的细胞学位置。 蛋白质和位置的任何变化，以及（4）还使用抗体来 用蛋白质印迹法检查任何化学修饰。 的 研究还将确定（5）分离是否可能是 受受精时产生或破坏的小分子影响， 通过富集内源性小分子的透化介质来评估 分子。 一个辅助但相关的目标是扩展新技术 用于通过使用笼状底物测量体内酶活性， 确定（6）氨基酸催化剂是否是重要的能量来源 促进发展。 所有的卵母细胞，即使是哺乳动物的卵母细胞，都有蛋黄， 这种蛋黄在支持早期发育中的作用还没有被认识到。 这种笼状基质的工作将扩展这种技术， 为发展的动力学提供新的见解。