Assay of Alkaline Phosphatase in Salmon Egg Cell Cytoplasm with Fluorescence Detection of Enzymatic Activity and Zinc Detection by ICP-MS in Relation to Metallomics Research
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- Hasegawa Takuya
- Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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- Sugita Masaru
- Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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- Takatani Kohei
- Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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- Matsuura Hirotaka
- Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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- Umemura Tomonari
- Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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- Haraguchi Hiroki
- Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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Abstract
Assay of alkaline phosphatase (ALP) in salmon egg cell cytoplasm was performed by size exclusion chromatography (SEC) with fluorescence detection of enzymatic activity and element-selective detection of zinc by ICP-MS, where 4-methylumbelliferyl phosphate (4-MUP) was used as the substrate for the enzymatic reaction of ALP. Two peaks were observed at retention times of ca. 21 and 27 min in the fluorescence-detected SEC chromatogram for salmon egg cell cytoplasm diluted 10-fold with 0.1 M Tris-HNO3 buffer solution, corresponding to the molecular weights (MWs) of ≥300 and 100 kDa, respectively. On the other hand, in the 66Zn-detected chromatogram for the same sample, a broad peak with a shoulder on the side of longer retention time was observed at ca. 21 min (MW≥300 kDa). When salmon egg cell cytoplasm was diluted 10-fold with pure water or 0.01 M Tris-HNO3 buffer solution, precipitation occurred. The supernatant obtained after filtration produced only one clear peak at a retention time of ca. 27 min in the fluorescence-detected chromatogram, at which the peak corresponding to zinc was also observed in the Zn-detected chromatogram. Therefore, the peak near 27 min was assigned to hydrophilic ALP in the salmon egg cell cytoplasm. Furthermore, the concentration of hydrophilic ALP (MW ca. 100 kDa) in the salmon egg cell cytoplasm was about 350 ng g−1, which was determined as an E. coli ALP-equivalent concentration by fluorescence intensity measurements.
Journal
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- Bulletin of the Chemical Society of Japan
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Bulletin of the Chemical Society of Japan 79 (8), 1211-1214, 2006
The Chemical Society of Japan
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Details 詳細情報について
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- CRID
- 1390001204143893248
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- NII Article ID
- 130004058746
- 10018210830
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- NII Book ID
- AA00580132
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- ISSN
- 13480634
- 00092673
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- NDL BIB ID
- 8009769
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed