Photometric assay for polynucleotide phosphorylase. 1999

L Fontanella, and S Pozzuolo, and A Costanzo, and R Favaro, and G Dehò, and P Tortora
Department of General Physiology and Biochemistry, Department of Genetics and Biology of Microorganisms, University of Milan, Via Celoria 26, Milano, I-20133, Italy.

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.

UI MeSH Term Description Entries
D010783 Photometry Measurement of the various properties of light. Photometries
D011117 Polyribonucleotide Nucleotidyltransferase An enzyme of the transferase class that catalyzes the reaction RNA(n+1) and orthophosphate to yield RNA(n) and a nucleoside diphosphate, or the reverse reaction. ADP, IDP, GDP, UDP, and CDP can act as donors in the latter case. (From Dorland, 27th ed) EC 2.7.7.8. Polynucleotide Phosphorylase,Nucleotidyltransferase, Polyribonucleotide,Phosphorylase, Polynucleotide
D011770 Pyruvate Kinase ATP:pyruvate 2-O-phosphotransferase. A phosphotransferase that catalyzes reversibly the phosphorylation of pyruvate to phosphoenolpyruvate in the presence of ATP. It has four isozymes (L, R, M1, and M2). Deficiency of the enzyme results in hemolytic anemia. EC 2.7.1.40. L-Type Pyruvate Kinase,M-Type Pyruvate Kinase,M1-Type Pyruvate Kinase,M2-Type Pyruvate Kinase,Pyruvate Kinase L,R-Type Pyruvate Kinase,L Type Pyruvate Kinase,M Type Pyruvate Kinase,M1 Type Pyruvate Kinase,M2 Type Pyruvate Kinase,Pyruvate Kinase, L-Type,Pyruvate Kinase, M-Type,Pyruvate Kinase, M1-Type,Pyruvate Kinase, M2-Type,Pyruvate Kinase, R-Type,R Type Pyruvate Kinase
D012015 Reference Standards A basis of value established for the measure of quantity, weight, extent or quality, e.g. weight standards, standard solutions, methods, techniques, and procedures used in diagnosis and therapy. Standard Preparations,Standards, Reference,Preparations, Standard,Standardization,Standards,Preparation, Standard,Reference Standard,Standard Preparation,Standard, Reference
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006593 Hexokinase An enzyme that catalyzes the conversion of ATP and a D-hexose to ADP and a D-hexose 6-phosphate. D-Glucose, D-mannose, D-fructose, sorbitol, and D-glucosamine can act as acceptors; ITP and dATP can act as donors. The liver isoenzyme has sometimes been called glucokinase. (From Enzyme Nomenclature, 1992) EC 2.7.1.1. Hexokinase A,Hexokinase D,Hexokinase II
D000244 Adenosine Diphosphate Adenosine 5'-(trihydrogen diphosphate). An adenine nucleotide containing two phosphate groups esterified to the sugar moiety at the 5'-position. ADP,Adenosine Pyrophosphate,Magnesium ADP,MgADP,Adenosine 5'-Pyrophosphate,5'-Pyrophosphate, Adenosine,ADP, Magnesium,Adenosine 5' Pyrophosphate,Diphosphate, Adenosine,Pyrophosphate, Adenosine
D016958 Pseudomonas putida A species of gram-negative, aerobic bacteria isolated from soil and water as well as clinical specimens. Occasionally it is an opportunistic pathogen.
D019298 Glucose-6-Phosphate An ester of glucose with phosphoric acid, made in the course of glucose metabolism by mammalian and other cells. It is a normal constituent of resting muscle and probably is in constant equilibrium with fructose-6-phosphate. (Stedman, 26th ed) Glucose 6 Phosphate

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