A novel fluorometric assay for ADP-ribose pyrophosphatase activity.
ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.
Song
Eun-Kyung
EK
Department of Microbiology and Immunology, Chonbuk National University Medical School, Chonju 561-182, South Korea.
Park
Hyung-Ju
HJ
Kim
Jong-Suk
JS
Lee
Hwang-Ho
HH
Kim
Uh-Hyun
UH
Han
Myung-Kwan
MK
D000244:Adenosine Diphosphate / Q000737:chemistry; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000378:metabolism; D001667:Binding, Competitive; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011755:Pyrophosphatases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012266:Ribose / Q000737:chemistry; D012267:Ribosemonophosphates / Q000737:chemistry; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D010710:Phosphates; D012267:Ribosemonophosphates; D000246:Adenosine Diphosphate Ribose; C031626:ribose-5-phosphate; D000244:Adenosine Diphosphate; D012266:Ribose; D000469:Alkaline Phosphatase; C403715:NUDT5 protein, human; D011755:Pyrophosphatases; C019620:ADPribose pyrophosphatase