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Development of a real-time method for detection of PPi hydrolysis. Jonas Eriksson*1; S Karamohamed2; P Nyrén1 (1) Dpt Biotechnology, RIT, Teknikringen 30-34, 100 44 Stockholm, Sweden; (2) Samer Karamohamed, Dpt Molecular and Cellular Biology, Harvard University, 7 Divinity Ave, Cambridge, MA 02138, USA, Tel: +1-617-4952301, samer@mcb.h      *(jonase@biochem.kth.se) A sensitive and simple method for real-time detection of enzymatic hydrolysis of inorganic pyrophosphate (PPi) has been developed. The method is based on enzymatic induced activation of the firefly luciferase activity in the presence of PPi. PPi inhibits the luciferase activity, but in the presence of a PPi hydrolysing enzyme the luciferase activity is restored and the luminescence output increases. The method was used to detect the hydrolytic activity of inorganic pyrophosphatase (PPase) (EC 3. 6. 1. 1) from Escherichia coli and Saccharomyces cerevisiae as well as alkaline phosphatase (AP) (EC 3. 1. 3. 1) from Shrimp. Depending on the properties of the enzyme under study either ATP or adenosine 5'-phosphosulfate (APS) can be used as substrate for the luciferase. This is an advantage for detection of PPi hydrolysis activity in crude extracts containing ATP hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of the activity of PPi hydrolyzing enzymes during different purification procedures. [Talk: eriksson.jonas.76162]

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