当前位置: X-MOL 学术Prog. Nucl. Magn. Reson. Spectrosc. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Studying enzymes by in vivo 13C magnetic resonance spectroscopy
Progress in Nuclear Magnetic Resonance Spectroscopy ( IF 6.1 ) Pub Date : 2009-10-01 , DOI: 10.1016/j.pnmrs.2009.06.002
Su Xu 1 , Jun Shen
Affiliation  

Magnetic resonance spectroscopy (MRS) allows noninvasive detection of specific biologically relevant molecules in vivo. It has become a very useful and versatile tool for both clinical and basic science studies because it can measure concentrations of many important endogenous and exogenous molecules such as the putative neuronal marker N-acetylaspartate [1], the 19F-containing selective serotonin reuptake inhibitor Prozac [2], glycogen[3], and adenosine triphosphate [4]. For an endogenous molecule, its concentration measured by MRS is usually the result of a complex balance among various metabolic fluxes with each of the fluxes controlled by a host of different enzymes. By introducing exogenous 13C-labeled substrates certain metabolic pathways can be measured using 13C MRS (e.g., the glutamate-glutamine cycling flux in the brain [5, 6]). In addition to concentrations and metabolic fluxes, an exceptional feature of MRS is its ability to measure the rate of an exchange reaction catalyzed by a specific enzyme in vivo using the technique of magnetization (or saturation) transfer. When kinetically relevant reporter molecules are spin labeled and spin transferred, their exchange rate can be quantified based on the competition between chemical exchange and the longitudinal relaxation time (T1). The theory of chemical exchange magnetization transfer was developed by chemists more than fifty years ago [7–12]. The phenomenon of in vivo enzyme-specific chemical exchange magnetization transfer was discovered approximately thirty years ago for adenosine triphosphate (ATP)-related exchange reactions [13] including the exchange reactions catalyzed by creatine kinase [14] and the invertebrate-originated arginine kinase [14]. The ability of noninvasively extracting information from specific enzymes using in vivo MRS is highly significant and has generated a great deal of enthusiasm [14–21]. In particular, creatine kinase-catalyzed magnetization transfer effect has been demonstrated to be a useful magnetic resonance reporter of gene expression [22]. Obviously, it would be highly desirable if more enzymes were accessible to in vivo MRS-based magnetization transfer spectroscopy methods. However, since the early discoveries of the above-mentioned enzymes involved in catalyzing the transfer of phosphate groups no new enzymes exhibiting detectable in vivo magnetization transfer effects had been found until our recent discovery of in vivo 13C magnetization transfer effects [23, 24]. Our interests in magnetization transfer started with the long-standing controversies on the rate of exchange between brain cytosolic glutamate/aspartate and mitochondrial α-ketoglutarate/oxaloacetate pools extracted from metabolic modeling of in vivo 13C MRS data. We hypothesized that if this exchange rate is very rapid it should be directly measurable using magnetization transfer. This line of research first led to the discovery of the in vivo magnetization transfer effect catalyzed by aspartate aminotransferase (AAT), then by lactate dehydrogenase (LDH) [25], malate dehydrogenase (MDH) [26], and carbonic anhydrase (CA) [27]. We demonstrated that the chemical exchange processes of these enzymes could be measured by 13C saturation transfer with 13C detection [24–27] and/or 13C saturation transfer with 1H detection techniques [28]. We also found that the exchange between 13C-labeled mitochondrial and cytosolic pools in brain is much faster than the tricarboxylic acid (TCA) cycle flux [29]. Here we endeavor to first give a brief overview of the early work in the field of in vivo 31P magnetization transfer spectroscopy because it is beyond the scope of this article to comprehensively review all in vivo magnetization transfer studies conducted on ATP-related enzymes using 31P MRS (the interested readers are referred to several excellent reviews on this topic [19–21]), Previous in vitro studies of enzyme systems using 13C NMR spectroscopy are also discussed. Then we will present the theoretical analyses and the experimental methods associated with detecting in vivo 13C magnetization transfer effects of a rapid chemical exchange process between small and large substrate pools, and review the current applications of in vivo 13C magnetization transfer spectroscopy to the study of enzymes. The chemical shifts of chemicals involved in enzyme-specific 13C magnetization transfer effects discovered so far are given in Table 1. Table 1 13C and 1H chemical shifts of molecules involved in enzyme-specific 13C magnetization transfer effects 2. Overview Creatine kinase (CK) has proven to be particularly amenable - in conjunction with 31P magnetization transfer spectroscopy - for elucidating rapid chemical exchange processes. CK catalyzes the phosphate of phosphocreatine (PCr) exchanges with the adenosine triphosphate (ATP) reaction, and is a key enzyme for maintaining cellular energy supplies:

中文翻译:

通过体内 13C 磁共振波谱研究酶

磁共振波谱 (MRS) 允许在体内无创检测特定的生物相关分子。它已成为临床和基础科学研究非常有用和通用的工具,因为它可以测量许多重要的内源性和外源性分子的浓度,例如推定的神经元标记 N-乙酰天冬氨酸 [1]、含 19F 的选择性血清素再摄取抑制剂百忧解[2]、糖原 [3] 和三磷酸腺苷 [4]。对于内源性分子,通过 MRS 测量的其浓度通常是各种代谢通量之间复杂平衡的结果,每个代谢通量由许多不同的酶控制。通过引入外源性 13C 标记的底物,可以使用 13C MRS 测量某些代谢途径(例如,大脑中的谷氨酸-谷氨酰胺循环通量 [5, 6])。除了浓度和代谢通量之外,MRS 的一个特殊特征是它能够使用磁化(或饱和)转移技术测量体内特定酶催化的交换反应的速率。当动力学相关的报告分子被自旋标记和自旋转移时,它们的交换率可以根据化学交换和纵向弛豫时间 (T1) 之间的竞争进行量化。化学交换磁化转移理论是五十多年前由化学家提出的 [7-12]。大约 30 年前,体内酶特异性化学交换磁化转移现象被发现用于三磷酸腺苷 (ATP) 相关的交换反应 [13],包括由肌酸激酶 [14] 和无脊椎动物来源的精氨酸激酶催化的交换反应 [13]。 14]。使用体内 MRS 从特定酶中非侵入性地提取信息的能力非常重要,并引起了极大的热情 [14-21]。特别是,肌酸激酶催化的磁化转移效应已被证明是一种有用的基因表达磁共振报告基因 [22]。显然,如果更多的酶可用于基于 MRS 的体内磁化转移光谱方法,那将是非常可取的。然而,由于参与催化磷酸基团转移的上述酶的早期发现,直到我们最近发现体内 13C 磁化转移效应 [23, 24] 之前,没有发现新的酶表现出可检测的体内磁化转移效应。我们对磁化转移的兴趣始于对从体内 13C MRS 数据的代谢建模中提取的脑细胞溶质谷氨酸/天冬氨酸和线粒体 α-酮戊二酸/草酰乙酸池之间交换率的长期争议。我们假设如果这种交换速度非常快,它应该可以使用磁化转移直接测量。这一系列研究首先发现了由天冬氨酸氨基转移酶 (AAT) 催化的体内磁化转移效应,然后是乳酸脱氢酶 (LDH) [25],苹果酸脱氢酶 (MDH) [26] 和碳酸酐酶 (CA) [27]。我们证明这些酶的化学交换过程可以通过 13C 检测的 13C 饱和转移 [24-27] 和/或 1H 检测技术的 13C 饱和转移 [28] 来测量。我们还发现大脑中 13C 标记的线粒体和细胞质池之间的交换比三羧酸 (TCA) 循环通量快得多 [29]。在这里,我们努力首先简要概述体内 31P 磁化转移光谱领域的早期工作,因为全面审查使用 31P MRS 对 ATP 相关酶进行的所有体内磁化转移研究超出了本文的范围。 (感兴趣的读者可以参考关于这个主题的几篇优秀评论[19-21]),还讨论了以前使用 13 C NMR 光谱对酶系统进行的体外研究。然后,我们将介绍与检测小型和大型底物池之间快速化学交换过程的体内 13C 磁化转移效应相关的理论分析和实验方法,并回顾体内 13C 磁化转移光谱在酶研究中的当前应用. 迄今为止发现的酶特异性 13C 磁化转移效应中涉及的化学物质的化学位移见表 1。 表 1 酶特异性 13C 磁化转移效应中涉及的分子的 13C 和 1H 化学位移 2. 概述 肌酸激酶 (CK) 具有被证明特别适合 - 结合 31P 磁化转移光谱 - 用于阐明快速化学交换过程。
更新日期:2009-10-01
down
wechat
bug