| Autor: |
Baykov AA; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia., Anashkin VA; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia., Malinen AM; Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland. |
| Jazyk: |
angličtina |
| Zdroj: |
Molecules (Basel, Switzerland) [Molecules] 2021 Apr 18; Vol. 26 (8). Date of Electronic Publication: 2021 Apr 18. |
| DOI: |
10.3390/molecules26082356 |
| Abstrakt: |
Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP i ) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP i as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP i synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP i hydrolysis, (b) continuous and fixed-time measurements of PP i synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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