JoséCarlos Cameselle

Specific ADP-ribose pyrophosphatase from Artemia cysts and rat liver: effects of nitroprusside, fluoride and ionic strength

One specific ADP-ribose pyrophosphatase (ADPRibase) has been identified in Artemia cysts, following a protocol that in rat liver allows the identification of three ADPRibases. Artemia ADPRibase resulted similar, but not identical, to rat liver ADPRibase-I with respect to known and novel properties disclosed in this work. In the presence of Mg2+, Artemia ADPRibase was highly specific for ADP-ribose and showed a low, 0.7 microM Km. Preincubation with the nitric oxide donor nitroprusside and dithiothreitol, elicited dose- and time-dependent, severalfold increase of Km and decrease of Vmax. At saturating ADP-ribose concentrations, fluoride was a strong inhibitor (IC50 approximately equal to 10-20 microM), whereas bringing ionic strength to 0.3-1.3 mol/l doubled the activity measured at lower or higher strengths. The novel fluoride and ionic strength effects were studied also with rat liver ADPRibase-I. Differences between the Artemia enzyme and ADPRibase-I concerned molecular weight (31,000 versus 38,500, respectively), Mn2+ ability to substitute for Mg2+ as the activating cation (better for the rat enzyme), and Vmax decrease by nitroprusside (not seen with the rat enzyme). The results are discussed in relation with the role of specific ADPRibases as protective factors limiting free ADP-ribose accumulation and protein glycation, and as targets for cytotoxic agents.

Enzyme saturation and inhibition kinetics studied from multiple progress curves recorded spectrophotometrically from single reaction mixtures for ADP-ribose pyrophosphatase

Saturation and inhibition kinetics data for rat liver ADP-ribose pyrophosphatase (EC 3.6.1.13) were obtained from progress curves initiated by the addition of substrate and recorded spectrophotometrically until the end point was reached. The hydrolysis of ADP-ribose was coupled to either alkaline phosphatase and adenosine deaminase or AMP deaminase. The validity of the approach was shown because: (i) the coupled hydrolysis of ADP-ribose was essentially irreversible; (ii) ADP-ribose pyrophosphate was stable at 37 degrees C in the conditions needed for the assay; and (iii) accumulated reaction products did not inhibit detectably in the conditions of the assay. In addition, several identical progress curves could be successively recorded by repetition of the addition of substrate. In that way it was possible to carry out complete inhibition studies by increasing the inhibitor concentration between successive substrate additions. Studying the inhibition by high D-ribose concentrations, meaningful results could be obtained at four different inhibitor concentrations in a single reaction mixture, which represented a great saving of enzyme preparation with respect to what would be needed in an equivalent initial rate study.

Interaction of divalent metal ions with fructose 2,6-bisphosphate and analogs

Abstract A study of the interaction of Mg(II), Ca(II), Zn(II), and Cd(II) ions with fructose phosphates like fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, and fructose 6-phosphate has been carried out. In order to perform this study, ionization constants of the ligands are determined from EMF data at 298 K and ionic strength I = 0.1 M in aqueous solution, and the stability constants of the complexes containing a 1:1 ratio of the ligand to the metal have been reported at 298 K.