Physiological And Metabolic Magnetic Resonance Studies

本项目研究化学毒素或物理因素如何改变代谢过程。NMR方法为完整系统、灌注器官、细胞悬液中的代谢和生理过程的研究以及细胞提取物的检查提供了独特的方法。在过去一年中，作为这项研究工作的一部分进行的主要研究概述如下： 项目一：硼酸盐与生物分子相互作用的表征对于理解硼的生理和毒理学性质以及硼酸盐配体的开发具有根本的重要性。 在过去的一年中，我们探索了硼酸盐可以作为磷酸盐模拟物的程度。 磷酸盐模拟的含氧阴离子，如钒酸盐，草酸盐，钨酸盐提供了基本的洞察磷转移的机制，也是重要的了解其毒理学和药理学性质。磷酸酯在细胞能量学、生物化学活化、信号转导和构象转换中起核心作用。硼酸根阴离子与磷酸根的结构相似性，结合其自发地使羟基和其他配体变性的能力，表明蛋白质上的磷酸酯识别位点可能对非酶促形成的硼酸酯表现出显着的亲和力。硼-11核磁共振研究和活性测量核糖核酸酶A在硼酸盐和几个胞苷类似物的存在下证明了一个稳定的三元RNase A3-脱氧胞苷-2-硼酸盐三元复合物的形成，模拟RNase A和2-胞苷一磷酸（2CMP）抑制剂之间形成的复合物。或者，对于三元RNA酶A、硼酸盐、2-脱氧胞苷混合物，没有观察到缓慢交换的硼酸盐共振，证明了2-羟基对于复合物形成的至关重要性。 滴定的三元复合物与2-CMP表明，它可以取代结合的硼酸酯的结合常数，这是接近报告的抑制常数RNase A的2CMP。 虽然随着pH的增加观察到三元络合物浓度的增加，但三元络合物的浓度并不像溶液中硼酸盐和硼酸单酯的浓度那样迅速增加。 该结果表明，酶对硼酸酯的亲和力随着pH的增加而降低，类似于对于磷酸酯抑制剂如2-或3-CMP的结合所观察到的pH依赖性。 也可以证明核糖核酸酶A与环状胞苷-2，3-硼酸酯的结合，该环状胞苷-2，3-硼酸酯是胞苷-2，3-环状磷酸底物的结构类似物。 这些结果与最近的报道一致，表明模拟相应磷酸酯的硼酸酯的原位形成可以支持酶催化。 项目2. 为了研究性别对心血管疾病的影响，我们利用了一个孤立的小鼠心脏模型，由于转基因品系的可用性和可用的NMR探针的小鼠心脏模型的大小兼容性。 我们以前的研究表明，在女性中看到的保护可能与心脏对底物选择的差异有关。 我们发现葡萄糖依赖性抑制多肽（GIP）保护男性心脏。 GIP处理还激活了许多与代谢相关的基因。 此外，使用13 C标记的底物，我们发现，相对葡萄糖/脂肪酸前体掺入乙酰辅酶A池在男性低于女性，GIP治疗消除了性别之间的差异。 我们现在试图测量丙酮酸脱氢酶（PDH）的活性。如果底物利用率存在差异，那么我们可以预期底物选择的这种变化通过PDH活性发生。 在另一项研究中，我们观察了硫氧还蛋白相互作用蛋白（Txnip）敲除小鼠。 Txnip通常被认为参与氧化还原调节。 使用31 P NMR，我们使用葡萄糖类似物2-脱氧葡萄糖监测心脏中的葡萄糖摄取。 我们发现Txnip-KO小鼠与对照组相比，葡萄糖摄取显著增加。 这一发现表明Txnip可能在心脏的代谢调节中发挥重要作用。 我们也研究了-arrestin基因敲除小鼠。β- 抑制蛋白是参与G-蛋白偶联信号传导的支架蛋白。 我们假设，这种蛋白质的损失可能会破坏预处理的保护作用发生的途径。 相反，我们发现预处理的保护作用仍然存在，尽管可能处于减弱的水平。

This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. The main studies performed as part of this research effort during the past year are summarized below: Project 1: Characterization of the interaction of borate with biological molecules is of fundamental importance for understanding the physiological and toxicological properties of boron, as well as for the development of boronate ligands. During the past year, we explored the extent to which borate can act as a phosphate mimic. Phosphate mimicry by oxyanions such as vanadate, molybdate, or tungstate have provided fundamental insight into the mechanisms of phosphotransfer, and also are important for understanding their toxicological and pharmacological properties. Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural analogy of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl and other ligands, suggested that phosphate-ester recognition sites on proteins might exhibit significant affinity for non-enzymatically formed borate esters. Boron-11 NMR studies and activity measurements on ribonuclease A in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A3-deoxycytidine-2-borate ternary complex that mimics the complex formed between RNase A and a 2-cytidine monophosphate (2CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2-deoxycytidine mixture, demonstrating the critical importance of the 2-hydroxyl group for complex formation. Titration of the ternary complex with 2-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant for RNase A by 2CMP. Although increased ternary complex concentration is observed with increasing pH, the concentration of ternary complex does not increase as rapidly as the concentrations of borate and borate monoesters in solution. This result indicates that the affinity of the enzyme for the borate ester decreases with increasing pH, similar to the pH dependence observed for the binding of phosphate ester inhibitors such as 2- or 3-CMP. RNase A binding of a cyclic cytidine-2,3-borate ester, which is a structural analog of the cytidine-2,3-cyclic phosphate substrate, could also be demonstrated. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters can support enzyme catalysis. Project 2. In order to study effects of gender on cardiovascular disease, we have utilized an isolated mouse heart model due to the availability of transgenic strains and to the size compatibility of the mouse heart model with available NMR probes. Our previous studies have suggested that the protection seen in females may be related to differences in substrate selection by the heart. We found that glucose-dependent inhibitory polypeptide (GIP) protected male hearts. GIP treatment also activated many genes associated with metabolism. Additionally, using 13C-labeled substrates, we found that the relative glucose/fatty acid precursor incorporation into the acetyl-CoA pool in males was lower than in females, and that GIP treatment abolished the difference seen between genders. We are now trying to measure the activity of pyruvate dehydrogenase (PDH). If there is a difference in substrate utilization, then we might expect this variation in substrate selection to occur through PDH activity. In another study, we looked at Thioredoxin-Interacting Protein (Txnip) knock out mice. Txnip is typically thought to be involved in redox regulation. Using 31P NMR, we monitored glucose uptake in the heart using the glucose analog 2-deoxyglucose. We found that the Txnip-KO mice exhibited dramatically increased glucose uptake compared to controls. This finding suggests that Txnip may play an important role in metabolic regulation of the heart. We also have looked at β-arrestin knock out mice. β-arrestin is a scaffolding protein involved in G-protein coupling signaling. We hypothesized that the loss of this protein could potentially disrupt the pathway through which the protective effect of preconditioning occurs. Instead, we found that protection from preconditioning was still present, though possibly at an attenuated level.