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首页> 美国卫生研究院文献>Biophysical Journal >Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.
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Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

机译：腺苷酸激酶及其同工型的细胞表征：双光子激发荧光成像和荧光相关光谱。

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Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.
机译：腺苷酸激酶（AK）是一种普遍存在的酶，可调节细胞中腺嘌呤核苷酸的稳态。来自鼠细胞的AK1beta（长型）与AK1（短型）具有相同的蛋白质序列，只是在其N端添加了18个氨基酸残基。假设这些残基充当蛋白质脂质修饰和蛋白质靶向质膜的信号。为了更好地了解这些AK同工型的细胞功能，我们使用了几种现代荧光技术来表征AK酶的这两种同工型。我们融合了胞质腺苷酸激酶（AK1）及其同工型（AK1beta）与增强的绿色荧光蛋白（EGFP），并在HeLa细胞中表达了嵌合蛋白。使用双光子激发扫描荧光成像，我们能够直接可视化活细胞中AK1-EGFP和AK1beta-EGFP的定位。 AK1beta-EGFP主要位于质膜上，而AK1-EGFP分布在整个细胞中，除了在核膜和某些囊泡中有痕量。我们在活细胞的特定域中进行了荧光相关光谱测量和光子计数直方图分析。对于AK1-EGFP，我们在细胞质中仅观察到一种扩散成分。对于AK1beta-EGFP，我们在质膜上观察到两个不同的扩散成分。一个对应于自由扩散蛋白，而另一个代表与膜结合的AK1beta-EGFP。相对于EGFP，AK1-EGFP的扩散速率降低了1.8倍，比我们预期的AK1-EGFP自由扩散的速率高50％。为了排除低聚物形成的可能性，我们进行了光子计数直方图分析，以直接分析AK1-EGFP和EGFP之间的亮度差异。根据我们的分析，我们得出结论，细胞质AK1-EGFP是单体的。荧光相关光谱被证明是定量研究靶蛋白在活细胞中迁移率的强大技术。这项技术在研究细胞中蛋白质相互作用和功能方面具有优势。

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