A simple sensitive fluorimetric assay of APS-kinase activity

Area of Science:

• Biochemistry
• Enzymology

Background:

• Adenosine phosphosulphokinase (APS-kinase) is crucial for synthesizing 3'-phosphoadenosine-5'-phosphosulfate (PAPS).
• PAPS is the primary sulfate donor in various biological sulfation reactions.
• Understanding APS-kinase kinetics is vital for comprehending cellular sulfate metabolism.

Purpose of the Study:

• To measure and characterize the activity of APS-kinase in different tissues.
• To determine the kinetic parameters (Km) for APS-kinase substrates and inhibitors.
• To establish a reliable assay for APS-kinase activity.

Main Methods:

• APS-kinase activity was indirectly assessed by measuring PAPS-dependent sulfotransferase activity using 4-methylumbelliferone (4-MU).
• Rat liver extract was treated with Sephadex G-25 and EDTA to remove Mg2+ and inhibit endogenous APS-kinase.
• Continuous fluorimetric assays were employed to quantify 4-MU-sulfate (4-MUS) formation.
• Kinetic analysis was performed to determine inhibition patterns and substrate affinities.

Main Results:

• APS-kinase activity was successfully measured using the described indirect assay.
• Substrate inhibition was observed at APS concentrations at and above 132 microM.
• Pyrophosphate (PP) was identified as an inhibitor of the APS-kinase reaction.
• Apparent Km values were determined: 14 microM for APS, 0.12 mM and 1.06 mM for ATP, and 0.09 mM for Mg2+.

Conclusions:

• The study established a functional assay for APS-kinase activity.
• Kinetic data reveal substrate inhibition by APS and inhibition by pyrophosphate.
• The determined kinetic parameters provide insights into the regulation of PAPS synthesis.