Class of 2015 Abstract / Objectives: Two characterized peptide substrates were assayed with human Polo-like kinase 4 to determine phosphorylation activity. A pilot library of Type-II kinase inhibitors designed to fit into the ATP-binding pocket will be screened to determine HsPlk4 inhibition activity, which will help characterize a novel drug compound.
Methods: Two peptide substrates of varying concentrations (2 uM, 1 uM, and 0.5 uM) were each combined with serial dilutions of HsPlk4 (1.25 uM, 0.625 uM, 0.313 uM, 0.156 uM, 0.078 uM, and 0.039 uM). EZ Reader detected phosphorylation activity by measuring fluorescence of both substrate and product, which separated at respective time points based on electrophoresis. The subsequent part of the experiment will be to inhibit the kinase activity with molecular inhibitors.
Results: The results showed HsPlk4 activity with the modified PLKtide, (5FAM)KKKTPSDSLYDDGLSKK(CONH2). All reactions with the various concentrations of substrate 1 and HsPlk4 showed phosphorylation activity. The reaction started within the first 10 minutes, quickly reaching maximal phosphorylation of substrate. No p-values were calculated due to lack of data.
Conclusions: No overall conclusions can be drawn based on the current results. Results showed the reaction reached its saturation point, so methods need to be refined to obtain data within the first 10 minutes. HsPlk4 phosphorylation of PLKtide confirmed the presumption that PLK family is a conserved family of Ser/Thr kinases. There are practical limitations for obtaining good kinetics data depicting enzyme activity, such as having EZ Reader quickly sample the reaction.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/614130 |
| Date | January 2015 |
| Creators | Annie Nguyen, Gokhale, Vijay, Rogers, Gregory |
| Contributors | Gokhale, Vijay, Rogers, Gregory, College of Pharmacy, The University of Arizona |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Report |
| Rights | Copyright © is held by the author. |
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