Real-time determination of the activity of ATPase by use of a water-soluble polythiophene
This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hyd...
| Main Authors: | , , , , , |
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| Format: | Journal Article |
| Language: | English |
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2013
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/98531 http://hdl.handle.net/10220/10754 |
| _version_ | 1826112914004639744 |
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| author | Umit, Hakan Yildiz Chia, Wei Sheng Diyar, Mailepessov Chia, Diana Xueqi Susana, Geifman Shochat Bo, Liedberg |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Umit, Hakan Yildiz Chia, Wei Sheng Diyar, Mailepessov Chia, Diana Xueqi Susana, Geifman Shochat Bo, Liedberg |
| author_sort | Umit, Hakan Yildiz |
| collection | NTU |
| description | This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K m and V max were 0.30 mmol L−1 ATP and 0.134 nmol ATP min−1 μg−1 enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ. |
| first_indexed | 2024-10-01T03:14:55Z |
| format | Journal Article |
| id | ntu-10356/98531 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:14:55Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | ntu-10356/985312020-03-07T12:18:20Z Real-time determination of the activity of ATPase by use of a water-soluble polythiophene Umit, Hakan Yildiz Chia, Wei Sheng Diyar, Mailepessov Chia, Diana Xueqi Susana, Geifman Shochat Bo, Liedberg School of Biological Sciences Centre for Biomimetic Sensor Science DRNTU::Science::Biological sciences This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K m and V max were 0.30 mmol L−1 ATP and 0.134 nmol ATP min−1 μg−1 enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ. 2013-06-27T01:57:58Z 2019-12-06T19:56:33Z 2013-06-27T01:57:58Z 2019-12-06T19:56:33Z 2012 2012 Journal Article Umit, H. Y., Chia, W. S., Diyar, M., Chia, D. X., Susana, G. S., & Bo, L. (2012). Real-time determination of the activity of ATPase by use of a water-soluble polythiophene. Analytical and Bioanalytical Chemistry, 404(8), 2369-2375. 1618-2642 https://hdl.handle.net/10356/98531 http://hdl.handle.net/10220/10754 10.1007/s00216-012-6341-8 en Analytical and bioanalytical chemistry © 2012 Springer-Verlag. |
| spellingShingle | DRNTU::Science::Biological sciences Umit, Hakan Yildiz Chia, Wei Sheng Diyar, Mailepessov Chia, Diana Xueqi Susana, Geifman Shochat Bo, Liedberg Real-time determination of the activity of ATPase by use of a water-soluble polythiophene |
| title | Real-time determination of the activity of ATPase by use of a water-soluble polythiophene |
| title_full | Real-time determination of the activity of ATPase by use of a water-soluble polythiophene |
| title_fullStr | Real-time determination of the activity of ATPase by use of a water-soluble polythiophene |
| title_full_unstemmed | Real-time determination of the activity of ATPase by use of a water-soluble polythiophene |
| title_short | Real-time determination of the activity of ATPase by use of a water-soluble polythiophene |
| title_sort | real time determination of the activity of atpase by use of a water soluble polythiophene |
| topic | DRNTU::Science::Biological sciences |
| url | https://hdl.handle.net/10356/98531 http://hdl.handle.net/10220/10754 |
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