著者

Saori TAKAHASHI Taku YOSHIYA Kumiko YOSHIZAWA-KUMAGAYE Toshihiro SUGIYAMA

出版者

Biomedical Research Press

雑誌

Biomedical Research (ISSN:03886107)

巻号頁・発行日

vol.36, no.3, pp.219-224, 2015-06-01 (Released:2015-06-24)

参考文献数

23

被引用文献数

37 53

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Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase which is highly homologous to angiotensin-converting enzyme (ACE). ACE2 produces vasodilator peptides angiotensin 1-7 from angiotensin II. In the present study, we synthesized various internally quenched fluorogenic (IQF) substrates (fluorophore-Xaa-Pro-quencher) based on the cleavage site of angiotensin II introducing N-terminal fluorophore N-methylanthranilic acid (Nma) and C-terminal quencher Nε-2,4- dinitrophenyl-lysine [Lys(Dnp)]. The synthesized mixed substrates “Nma-Xaa-Pro-Lys(Dnp)” were hydrolyzed by recombinant human (rh) ACE2. The amount of each product was determined by liquid chromatography mass spectrometry (LC-MS) with fluorescence detection and it was found that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2. The Km, kcat, and kcat/Km values of Nma-His-Pro-Lys(Dnp) on rhACE2 were determined to be 23.3 μM, 167 s−1, and 7.17 μM−1 s−1, respectively. Using the rhACE2 and the newly developed IQF substrate, we found rhACE2 inhibitory activity in soybean and isolated the active compound soybean ACE2 inhibitor (ACE2iSB). The physicochemical data on the isolated ACE2iSB were identical to those of nicotianamine. ACE2iSB strongly inhibited rhACE2 activity with an IC50 value of 84 nM. This is the first demonstration of an ACE2 inhibitor from foodstuffs.

著者

Tomoyo Nishihira Asami Miyano Takayuki Ohnuma Takeshi Gotoh Saori Takahashi Kazue Narihiro Kazuhiko Yamashita Tamo Fukamizo

出版者

日本応用糖質科学会

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

vol.61, no.4, pp.113-116, 2014 (Released:2014-11-20)

参考文献数

16

被引用文献数

2

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A simple turbidimetric assay using chitin nanofiber as the substrate was employed to measure chitinase activity. The higher dispersive properties of chitin nanofibers enabled the degradation of chitin to be monitored turbidimetrically. When non-processive chitinases, a family GH18 chitinase from the tobacco plant and a GH19 chitinase from rye seeds, were added to the β-chitin nanofiber suspension, no significant changes were observed in the turbidity of the suspension, however, the amounts of reducing sugars produced were significantly high and small amounts of GlcNAc and (GlcNAc)2 were detected by HPLC in the reaction mixture. However, the addition of a processive family GH18 chitinase, Serratia marcescence chitinase B or chitinase from Autographa californica multiple nucleopolyhedrovirus, resulted in a significant decrease in the turbidity of the chitin nanofiber suspension, and produced larger amounts of reducing sugars including GlcNAc and (GlcNAc)2. The rate of decreases in turbidity was clearly dependent upon the enzyme concentration. We concluded that the turbidimetric assay using β-chitin nanofibers as the substrate was useful for measuring the activities of processive chitinases.