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http://223.31.159.10:8080/jspui/handle/123456789/1027
Title: | Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+ -enhanced kinase activity in vitro |
Authors: | Chauhan, Sujata Samanta, Subhasis Sharma, Nitin Thakur, Jitendra K. Dev, Kamal Sourirajan, Anuradha |
Keywords: | Biological sciences Cell biology Proteins Biochemistry Cancer research Health sciences Oncology PLK Cdc5 Saccharomyces cerevisiae GST-Cdc5 In vitro kinase assay Mg2+ |
Issue Date: | 2019 |
Publisher: | Elsevier B.V. |
Citation: | Heliyon 5(12): e03050 |
Abstract: | Phosphorylation of proteins on serine/threonine residues represents an important biochemical mechanism to regulate several cellular processes. Polo-like kinases (PLKs) are a family of serine-threonine kinases that play an imminent role in cell cycle regulation in yeast to humans, and thus an important therapeutic target for cancers. The present study provides insights into the enzymatic features of Saccharomyces cerevisiae PLK, Cdc5 using in vitro casein phosphorylation assays. The recombinant yeast PLK, GST-Cdc5 showed maximum casein phosphorylation activity at 30 °C, pH 9 and 45 min of incubation period. GST-Cdc5 exhibited a KM of 1.35 μM for casein, and high affinity for ATP, since addition of non-radioactive ATP chased out casein phosphorylation by radiolabeled ATP. The recombinant enzyme showed maximum kinase activity at 2.7 μM of GST-Cdc5. Casein was found to be the best in vitro substrate of GST-Cdc5 followed by BSA (Bovine Serum Albumin) and MBP (Myelin Basic Protein). Of the metal ions tested, Mg2+ (at 20 mM) was found to enhance GST-Cdc5 kinase activity, while Ca2+ (at 5 mM) and Mn2+ (at 10 mM) inhibited the same. The presence of EDTA, SDS and PMSF inhibited phosphorylation by GST-Cdc5, while DTT had no effect. The recombinant GST-Cdc5 can be used as a tool for deciphering PLKs’ structure and functions, which are still at infancy. |
Description: | Accepted date: 11 December 2019 |
URI: | http://223.31.159.10:8080/jspui/handle/123456789/1027 |
ISSN: | 2405-8440 |
Appears in Collections: | Institutional Publications |
Files in This Item:
File | Description | Size | Format | |
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Thakur JK_2019_3.pdf | 1.78 MB | Adobe PDF | ![]() View/Open |
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