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排序方式: 共有475条查询结果,搜索用时 93 毫秒
471.
Takahito Furuya Issey Takehara Asuka Shimura Hisanao Kishimoto Tomoya Yasujima Kinya Ohta Yoshiyuki Shirasaka Hiroaki Yuasa Katsuhisa Inoue 《Biochemical and biophysical research communications》2018,495(3):2152-2157
Bioluminescence (BL) imaging based on d-luciferin (d-luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc–luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293?cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293?cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23?μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent Km of 0.36?μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. 相似文献
472.
Identification of Brassinosteroids That Appear to Be Derived from Campesterol and Cholesterol in Tomato Shoots 总被引:4,自引:0,他引:4
To obtain information about the biosynthesis of brassinosteroids(BRs) in tomato shoots, we examined endogenous BRs by gas chromatography-massspectrometry. We identified two C28 BRs, namely, castasteroneand 6-deoxocastasterone, and a C27 BR, 28-norcastasterone. Ourfindings suggest that the major BRs in tomato are derived fromcampesterol and cholesterol.
4Present address: National Research Institute of Vegetables,Ornamental Plants and Tea, 360 Kusawa, Ano, Mie, 514-23 Japan. 相似文献
473.
K Kohama T Okagaki K Hayakawa Y Lin R Ishikawa T Shimmen A Inoue 《Biochemical and biophysical research communications》1992,184(3):1204-1211
The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables. 相似文献
474.
Yoshiyuki Sasajima Yusuke Kohama Miki Kojima-Misaizu Naoya Kurokawa Yuko Hara Jinhua Dong Masaki Ihara Hiroshi Ueda 《Molecular biotechnology》2014,56(10):953-961
Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75 °C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization. 相似文献
475.