I am trying to see the enzymatic activity of a specific enzyme involved in nucleotide biosynthesis. Does anyone have any suggestion about PPAT enzymatic assay?
Dominique Liger
Hi there,
The actual reaction is the following: PRPP+Glutamine gives PRA(phosporibosyl-amine)+Glutamate+PPi
Therefore an easy method to measure such activity is to couple the assay with Glutamate deshydrogenase in presence of NAD+. The reaction is followed by observing the increase of OD at 340nm (formation of NADH).
An other method is to measure PPi formation via PPase and phosphate determination.
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Miguel de abreu
Just as Dominique described, I use malachite green method described in Analytical Biochemistry 171,266-270 (1988) to determine inorganic phosphate
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