A colorimetric method for the assay of ADP-glucose pyrophosphorylase

CORINA FUSARI; ANA M. DEMONTE; CARLOS M. FIGUEROA; MABEL ALEANZI; ALBERTO A. IGLESIAS

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2006 vol. 352 p. 145 - 147

0003-2697

The purpose of the current work was to develop a relatively simple method to assay ADPGlcPPase activity having high sensitivity, accuracy, and reliability. We sought a procedure allowing the assay in both of the reaction directions, based on a technique suitable for the screening of numerous samples. The method quantifies inorganic orthophosphate released from the specific hydrolysis of the enzyme activity products. For ADPGlc synthesis, the method measures Pi after hydrolysis of PPi by inorganic pyrophosphatase. Pyrophosphorolysis is assayed by determining Pi derived from CF1-ATPase-mediated hydrolysis of ATP. Pi dosage is performed by the technique based in the formation of a phosphomolybdate-Malachite Green complex. We optimized the procedure to a microscale grade, reaching convenient sensitivity and the possibility of automation, by using a microplate (multiwell) absorbance reader.