María Jesús Costas

A specific, low Km ADP-ribose pyrophosphatase from rat liver

Two rat liver ADP‐ribose pyrophosphatases (ADPRibases) were partially purified. ADPRibase‐I hydrolyzed ADP‐ribose (K m=0.5 μM) giving AMP as a product, required Mg2+ or, less efficiently, Mn2+ (Ca2+ was not active), its activity changed little between pH 7 and 9, and was specific for ADP‐ribose as it did not hydrolyze ADP‐glucose, NAD+, NADH or diadenosine 5′,5″‐P 1,P n ‐n‐phosphates (Ap2A, Ap3A). ADPRibase‐II showed similar properties, except that the K m for ADP‐ribose was 50 μM and may be non‐specific, as the same preparation hydrolyzed ADP‐glucose, NADH and Ap2A. ADPRibase‐I fulfils the requirements of a specific turnover pathway consistent with a cellular role for free ADP‐ribose.

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.

Diadenosine tetraphosphate activates AMP deaminase from rat muscle

Diadenosine tetraphosphate, Ap4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap4A appeared to induce Michaelian kinetic behavior. The activation by Ap4A was not dependent on the presence of either MgCl2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.

Dinucleosidetetraphosphatase inhibition by Zn(II)

Almost complete inhibition of partially purified dinucleoside-tetraphosphatase (EC 3. 6. 1. 17) was observed with 5 microM Zn(II). The inhibition was reversed by EDTA and was time dependent, reaching a maximum after 5 min of incubation at 37 degrees C. Zn(II) behaved as a non-competitive inhibitor of the reaction, leaving unaltered the Km value for the enzyme towards diadenosine tetraphosphate. The cellular level of this compound may be directly related to the Zn(II) content since, besides the inhibition here described, Zn(II) has been reported by others to be an activator of the synthesis of diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-t RNA synthetases.

Glycine-enhanced inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase-I by EDTA: a full account of the reported inhibition by commercial preparations of acidic fibroblast growth factor (FGF-1).

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture‐grade acidic fibroblast growth factor (FGF‐1) correlates with a low‐M r contaminant. 1H‐NMR analyses revealed EDTA in the total‐volume fractions of a gel‐filtration experiment, where all the inhibitory activity of the FGF‐1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF‐1 or the EDTA‐containing fractions) was time‐dependent, blocked by the substrate p‐nitrophenyl‐dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF‐1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP‐ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP‐ribose, form and hydrolyze the N 1‐glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP‐ribose/CDP‐alcohol pyrophosphatase (ADPRibase‐Mn) and found that cADPR is an ADPRibase‐Mn ligand and substrate. ADPRibase‐Mn activity on cADPR was 65‐fold less efficient than on ADP‐ribose, the best substrate. This is similar to the ADP‐ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase‐Mn was N 1‐(5‐phosphoribosyl)‐AMP, suggesting a novel route for cADPR turnover.

ADP‐ribose pyrophosphatase‐I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol

Free ADP‐ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low‐Km ADP‐ribose pyrophosphatase‐I (ADPRibase‐I) is well suited to control free ADP‐ribose and nonenzymatic ADP‐ribosylation. In vitro, the acetaminophen metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) decreases ADPRibase‐I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase‐I in vivo. Rats pretreated with 3‐methylcholanthrene and L‐buthionine‐[S,R]‐sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/kg; n = 5) or vehicle (n = 3). ADPRibase‐I partially purified from acetaminophen‐overdosed rats showed a decreased Vmax (0.32 ± 0.09 versus 0.60 ± 0.03 mU/mg of liver protein; p < 0.01) not reverted by DTT and an increased Km for ADP‐ribose (1.39 ± 0.31 versus 0.67 ± 0.05 μM; p < 0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase‐I from normal rat liver with oxidized glutathione elicited a time‐ and dose‐dependent, DTT‐reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase‐I can be regulated by thiol exchange and that the increase of Km elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP‐ribose concomitant to ADPRibase‐I inhibition could contribute to the hepatotoxicity of acetaminophen.

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.

Occurrence of dinucleosidetriphosphatase in the cytosol and particulate fractions from rat liver

Dinucleosidetriphosphatase (EC 3.6.1.29) is present in both the 37,000 g rat liver supernatant and precipitate (50 mU/g each fraction). These two activities show matching molecular weights, isoelectric points, substrate specificities, Km values, bivalent cation requirements and inhibition by zinc (II). The particulate triphosphatase and a residual dinucleosidetetraphosphatase (EC 3.6.1.17) are solubilized by freeze-thawing or by Triton X-100. Detergent treatment also extracts an unspecific phosphodiesterase I activity (EC 3.1.4.1) which also splits dinucleoside polyphosphates. The above findings suggest the occurrence of cytosolic and particulate degradative pathways for dinucleoside polyphosphates.

Rat liver ADP-ribose pyrophosphatase-I as an in vitro target of the acetaminophen metabolite N-acetyl-p-benzoquinoneimine

N-acetyl-p-benzoquinoneimine (NAPQI) is the metabolite responsible for acetaminophen hepatotoxicity. ADP-ribose pyrophosphatase-I (ADPRibase-I; EC 3.6.1.13) hydrolyzes protein-glycating ADP-ribose. The results show NAPQI-dependent alterations of ADPRibase-I leading to strong inhibition: a fast Km increase produced by low concentrations, and a time-dependent Vmax decrease by higher NAPQI concentrations. Both effects were prevented by thiols, but not reverted by them, nor by gel filtration of NAPQI-treated enzyme. Liver ADPRibase-I can be a target of NAPQI-dependent arylation. The inhibition or inactivation of the enzyme would contribute to increasing the free ADP-ribose concentration and nonenzymatic ADP-ribosylation, which is coherent with results linking free ADP-ribose-producing pathways to acetaminophen toxicity.