A highly sensitive, colorimetric method for the assay of ADP-glcucose pyrophosphorylase activity

FUSARI C; DEMONTE A; FIGUEROA C; ALEANZI M; IGLESIAS AA

Villa Carlos Paz

Congreso; XXXVIII Reunión Anual de la SAIB; 2002

SAIB

ADPglucose pyrophosphorylase (ADPGlcPPase) reversibly catalyzes the synthesis of ADPGlc and PPi from ATP and Glc1P. The enzyme plays a key role in the biosynthesis of glycogen in bacteria and starch in plants, since the production of ADPGlc is the limiting step for polysaccharide accumulation in these organisms. Highly sensitive assays of ADPGlcPPase activity are performed using radioactive Glc1P or PPi. An alternative previously described spectrophotometric method is limited because: i) its relative low sensitivity, and ii) it is useful only to measure pyrophosphorolysis of ADPGlc. We developed a highly sensitive, colorimetric microassay based in the detection of Pi, utilizing the principle that at low pH Malachite Green forms a complex with phosphomolybdate with a marked shift in absorption maximum and a high molar absorption coefficient. In the ADPGlc synthesis direction, the microassay detects production of PPi after its hydrolysis by an excess of coupled inorganic pyrophosphatase. Under the assay conditions, the curve for Pi detection at 650 nm was linear (R = 0.99) between 0.5-5.0 nmol. Assays of ADPGlcPPase activity using partially purified enzyme exhibited a high correlation with measurements performed using the radiochemical method. The colorimetric assay is also useful to assay the enzyme in the direction of pyrophosphorolysis of ADPGlc; thus, constituting a highly sensitive and reliable technique for the kinetic analysis of ADPGlcPPase.