MITOCHONDRIA FROZEN WITH TREHALOSE RETAIN BIOLOGICAL FUNCTIONS

用海藻糖冷冻线粒体保留生物功能

• 批准号: 7601060
• 负责人: DONALD DAVID NEWMEYER
• 金额: $ 1.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601060
• 关键词: ATP Synthesis Pathway; Apoptosis; Bax protein; Biological Process; Buffers; Calcium; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Equilibrium; Freezing; Funding; Grant; In Vitro; Institution; Liver Mitochondria; Mannitol; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Mus; Organelles; Outer Mitochondrial Membrane; Process; Protein Family; Protein Import; Proteins; Research; Research Personnel; Resources; Source; Standards of Weights and Measures; Sucrose; Swelling; Trehalose; United States National Institutes of Health; cytochrome c; pro-apoptotic protein; sugar

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In apoptosis, the Bcl-2 family proteins Bax and Bak disrupt the normal barrier function of the mitochondrial outer membrane (MOM), causing pro-apoptotic proteins such as cytochrome c, Omi and Smac to be released from the intermembrane space (IMS) into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria maintained in a physiologically balanced buffer. The MOMs of these organelles remain intact, unless Bak is activated, e.g. by incubation with a BH3-only protein such as tBid. In contrast, the MOMs of mitochondria frozen/thawed in standard sucrose-mannitol buffers immediately become leaky, rendering them useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained their MOM integrity and responsiveness to BH3-only proteins, much like fresh mitochondria. Freezing in trehalose buffer also preserved biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential and mitochondrial protein import. The ability to store active mitochondria will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在细胞凋亡中，Bcl2家族蛋白Bax和Bak破坏了线粒体外膜的正常屏障功能，导致细胞色素c、Omi和Smac等促凋亡蛋白从膜间隙释放到细胞质中。这一过程可以在体外研究，新鲜分离的小鼠肝脏线粒体保持在生理平衡的缓冲液中。这些细胞器的母细胞保持完好，除非Bak被激活，例如通过与仅BH3蛋白孵育，如TBID。相比之下，在标准的蔗糖-甘露醇缓冲液中冷冻/解冻的线粒体的母亲立即变得泄漏，使它们对细胞凋亡研究毫无用处。然而，我们在这里展示了冷冻在含有糖和海藻糖的缓冲液中的线粒体，保持了它们母亲的完整性和对仅BH3蛋白的反应，很像新鲜的线粒体。在海藻糖缓冲液中冷冻也保存了ATP合成、钙诱导肿胀、跨膜电位和线粒体蛋白输入等生物学功能。储存活性线粒体的能力将有助于研究细胞凋亡和其他依赖于完整的MOM的线粒体功能。