RAT-LIVER NUCLEOSIDE DIPHOSPHOSUGAR OR DIPHOSPHOALCOHOL PYROPHOSPHATASES DIFFERENT FROM NUCLEOTIDE PYROPHOSPHATASE OR PHOSPHODIESTERASE-I - SUBSTRATE SPECIFICITIES OF MG2+-DEPENDENT AND OR MN2+-DEPENDENT HYDROLASES ACTING ON ADP-RIBOSE

Three rat liver nucleoside(5') diphosphosugar (NDP-sugar) or nucleoside(5') diphosphoalcohol pyrophosphatases are described: two were previously identified in experiments measuring Mg2+-dependent ADP-ribose pyrophosphatase activity (Miro et al. (1989) FEBS Lett. 244, 123-126), and the other is a new, Mn2+-dependent ADP-ribose pyrophosphatase. They are resolved by ion-exchange chromatography, and differ by their substrate and cation specificities, K-M, values for ADP-ribose, pH-activity profiles, molecular weights and isoelectric points. The enzymes were tested for activity towards: reducing (ADP-ribose, IDP-ribose) and non-reducing NDP-sugars (ADP-glucose, ADP-mannose, GDP-mannose, UDP-mannose, UDP-glucose, UDP-xylose, CDP-glucose), CDP-alcohols (CDP-glycerol, CDP-ethanolamine, CDP-choline), dinucleotides (diadenosine pyrophosphate, NADH, NAD(+), FAD), nucleoside(5') mono- and diphos- phates (AMP, CMP, GMP, ADP, CDP) and dTMP p-nitrophenyl ester. Since the enzymes have not been purified to homogeneity, more than three pyrophosphatases may be present, but the co-purification of activities, thermal co-inactivation, and inhibition experiments give support to: (i) an ADP-ribose pyrophosphatase highly specific for ADP(IDP)-ribose in the presence of Mg2+, but active also on non-reducing ADP-hexoses and dinucleotides (not on NAD(+)) when Mg2+ was replaced with Mn2+; (ii) a Mn2+-dependent pyrophosphatase active on ADP(IDP)-ribose, dinucleotides and CDP-alcohols; (iii) a rather unspecific pyrophosphatase that, with Mg2+, was active on AMP(IMP)-containing NDP-sugars and dinucleotides (not on NAD(+)), and with Mn2+, was also active on non-adenine NDP-sugars and CDP-alcohols. The enzymes differ from nucleotide pyrophosphatase/phosphodiesterase-I (NPPase/PDEaseI) by their substrate specificities and by their cytosolic location and solubility in the absence of detergents. Although NPPase/PDEaseI is much more active in rat liver, its known location in the non-cytoplasmic sides of plasma and endoplasmic reticulum membranes, together with the known cytoplasmic synthesis of NDP-sugars and CDP-alcohols, permit the speculation that the pyrophosphatases studied in this work may have a cellular role.