Abstract
Determining enzyme activities involved in photorespiration, either in a crude plant tissue extract or in a preparation of a recombinant enzyme, is time-consuming, especially when large number of samples need to be processed. This chapter presents a phosphoglycolate phosphatase (PGLP) activity assay that is adapted for use in a 96-well microplate format. The microplate format for the assay requires fewer enzymes and reagents and allows rapid and less expensive measurement of PGLP enzyme activity. The small volume of reaction mix in a 96-well microplate format enables the determination of PGLP enzyme activity for screening many plant samples, multiple enzyme activities using the same protein extract, and/or identifying kinetic parameters for a recombinant enzyme. To assist in preparing assay reagents, we also present an R Shiny buffer preparation app for PGLP and other photorespiratory enzyme activities and a Km and Vmax calculation app.
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Acknowledgments
This work was supported by the US Department of Energy Office of Science, Basic Energy Sciences under Award DE- FG02-91ER20021 and the National Science Foundation under grant number 2030337. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231.
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Roze, L.V., Johnson, A., Gregory, L.M., Tejera-Nieves, M., Walker, B.J. (2024). High Throughput Phosphoglycolate Phosphatase Activity Assay Using Crude Leaf Extract and Recombinant Enzyme to Determine Kinetic Parameters Km and Vmax Using a Microplate Reader. In: Walker, B.J. (eds) Photorespiration. Methods in Molecular Biology, vol 2792. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3802-6_1
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DOI: https://doi.org/10.1007/978-1-0716-3802-6_1
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