Photometric assay for polynucleotide phosphorylase.
Polynucleotide phosphorylase (PNPase) is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of NDPs. It is also believed to play a key role in turnover of prokaryotic transcripts, thus regulating gene expression. At the moment, only radioisotopic methods are available for assaying PNPase in crude extracts; these involve incubating [32P]phosphate and poly(A) in the presence of the enzyme, separating [32P]phosphate from [32P]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 present 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-phosphate is then revealed by a glucose-6-phosphate dehydrogenase reaction. Based on 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 PNPase activity. Experimental data confirmed the theoretical predictions and showed that the assay was quantitative and unquestionably specific. We also devised a simple procedure for determining absolute enzyme activities (expressed in micromoles of product formed per minute) using exact amounts of pure PNPase as internal standards.
Fontanella
L
L
Department of General Physiology and Biochemistry, Department of Genetics and Biology of Microorganisms, University of Milan, Via Celoria 26, Milano, I-20133, Italy.
Pozzuolo
S
S
Costanzo
A
A
Favaro
R
R
Dehò
G
G
Tortora
P
P
D000244:Adenosine Diphosphate; D004926:Escherichia coli / Q000201:enzymology; D019298:Glucose-6-Phosphate; D006593:Hexokinase; D010783:Photometry / Q000379:methods* / Q000592:standards; D011117:Polyribonucleotide Nucleotidyltransferase / Q000032:analysis* / Q000592:standards; D016958:Pseudomonas putida / Q000201:enzymology; D011770:Pyruvate Kinase; D012015:Reference Standards
D019298:Glucose-6-Phosphate; D000244:Adenosine Diphosphate; D006593:Hexokinase; D011770:Pyruvate Kinase; D011117:Polyribonucleotide Nucleotidyltransferase