Photometric assay for polynucleotide phosphorylase

Polynucleotide phosphorylase (PNPase) is a prokaryotic enzyme that catalyze s phosphorolysis of polynucleotides with release of NDPs. It is also believ ed to play a key role in turnover of prokaryotic transcripts, thus regulati ng gene expression. At the moment, only radioisotopic methods are available for assaying PNPase in crude extracts; these involve incubating [P-32]phos phate and poly(A) in the presence of the enzyme, separating (P-32)phosphate from [P-32]ADP, and quantifying ADP by scintillation counting. Photometric assay using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes is not feasible in crude extracts because of endogenous ATPase activities, which regenerate ADP from the ATP released by pyruvate kinase. Here, we pr esent a simple photometric assay that uses a cyclic detection system which, due to the sequential action of pyruvate kinase and hexokinase, results in an exponential increase of ADP and glucose 6-phosphate. Glucose 6-phosphat e is then revealed by a glucose-6-phosphate dehydrogenase reaction. Based o n the theoretical model, a linear increase in absorbance is predicted as a function of the square of the reaction time, with a slope proportional to P NPase activity. Experimental data confirmed the theoretical predictions and showed that the assay was quantitative and unquestionably specific. We als o devised a simple procedure for determining absolute enzyme activities (ex pressed in micromoles of product formed per minute) using exact amounts of pure PNPase as internal standards. (C) 1999 Academic Press.