Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

作者：Pieslinger Anja Maria; Hoepflinger Marion Christine; Tenhaken Raimund^*

来源：Analytical Biochemistry, 2009, 388(2): 254-259.

DOI：10.1016/j.ab.2009.03.002

• Lilium longiflorum
• Pollen glucuronokinase
• HPLC
• Ion-exchange chromatography
• UDP-glucuronic acid pyrophosphorylase
• UDP-glucuronic acid
• Sugar-1-kinase

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-Sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a Coupling enzyme. Glucuronokinase was partially purified on a DEAF-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is Used by UDP-Sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-Sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange Column or a reversed phase Cl 8 Column at a wavelength of 260 nm. This assay is adaptive to different kinases and Sugars because of the broad Substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min(-1). Furthermore, it can be used for determination of pure kinases as well as crude OF partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.

• 出版日期2009-5-15