Rat liver nucleoside diphosphosugar or diphosphoalcohol pyrophosphatases different from nucleotide pyrophosphatase or phosphodiesterase I: substrate specificities of Mg 2+- and/or Mn 2+-dependent hydrolases acting on ADP-ribose

Abstract

Three rat liver nucleoside(5′) diphosphosugar (NDP-sugar) or nucleoside(5′) diphosphoalcohol pyrophosphatases are described: two were previously identified in experiments measuring Mg 2+-dependent ADP-ribose pyrophosphatase activity (Miró et al. (1989) FEBS Lett. 244, 123–126), and the other is a new, Mn 2+-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 diphosphates (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 Mg 2+, but active also on non-reducing ADP-hexoses and dinucleotides (not on NAD +) when Mg 2+ was replaced with Mn 2+; (ii) a Mn 2+-dependent pyrophosphatase active on ADP(IDP)-ribose, dinucleotides and CDP-alcohols; (iii) a rather unspecific pyrophosphatase that, with Mg 2+, was active on AMP(IMP)-containing NDP-sugars and dinucleotides (not on NAD +), and with Mn 2+, 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.