Rat liver nucleoside diphosphosugar or diphosphoalcohol pyrophosphatases different from nucleotide pyrophosphatase or phosphodiesterase I: substrate specificities of Mg2+- and/or Mn2+-dependent hydrolases acting on ADP-ribose

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

“…A similar activity has also been reported in embryonic cysts of the brine shrimp Artemia franciscana [25]. The cytosolic Mn# + -dependent ADP-ribase has no activity at all with Mg# + ions [16]. Therefore YSA1H does not seem to correspond to any of these enzymes.…”

“…The cytosolic ADP-ribase I and the mitochondrial enzyme are very similar in terms of low (micromolar) K m for ADP-ribose and a stringent substrate specificity limited to ADP-ribose and IDP-ribose [16,24]. A similar activity has also been reported in embryonic cysts of the brine shrimp Artemia franciscana [25].…”

“…Substrates were screened by measuring the P i released in a coupled assay by co-incubation of substrate with YSA1H protein and either alkaline phosphatase or inorganic pyrophosphatase [16,18]. The standard assay (200 µl) for phosphodiester substrates was incubation for 10 min at 37 mC with 50 mM Tris\HCl (pH 7.5)\5 mM MgCl # \1 mM dithiothreitol\0.3 mM substrate\ 0.25 µg of Trx-YSA1H fusion protein and 0.5 µg (1 unit) of alkaline phosphatase.…”

“…Comparative data are also presented in Table 1 for the human erythrocyte ADP-ribose pyrophosphatase, rat liver ADPribase II and calf liver ADP-sugar hydrolase. When assayed with 5 mM Mn# + in place of Mg# + , rat liver ADP-ribase II shows greatly increased activity with UDP-sugars and CDP-sugars and CDP-alcohols [16]. YSA1H activity towards CDP-glycerol was increased 4-fold when Mn# + replaced Mg# + , in comparison with the 50-fold increase observed with ADP-ribase II, whereas the activity of YSA1H towards ADP-sugars was decreased by 50-70 % with Mn# + .…”

“…The elimination of free ADP-ribose therefore seems to fall within the category of ' housecleaning ' functions proposed for the MutT motif family. An ADP-ribose pyrophosphatase has recently been isolated from human erythrocytes [12,15], whereas three cytoplasmic and one mitochondrial ADP-ribose pyrophosphatases in rat liver have been described [16]. A possibly distinct ADP-sugar pyrophosphatase (EC 3.6.1.21) has also been isolated from mammalian liver [17].…”