Characterisation of a bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) from Drosophila melanogaster.
The intracellular functions of diadenosine polyphosphates are still poorly defined. To understand these better, we have expressed and characterized a heat stable, 16.6kDa Nudix hydrolase (Apf) that specifically metabolizes these nucleotides from a Drosophila melanogaster cDNA. Apf always produces an NTP product, with substrate preference depending on pH and divalent ion (Zn(2+) or Mg(2+)). For example, diadenosine tetraphosphate is hydrolysed to ATP and AMP with K(m), k(cat) and k(cat)/K(m) values 9microM, 43s(-1) and 4.8microM(-1)s(-1) (pH 6.5, 0.1mMZn(2+)) and 12microM, 13s(-1) and 1.1microM(-1)s(-1) (pH 7.5, 20mMMg(2+)), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20mMMg(2+) (K(m), k(cat) and k(cat)/K(m) values of 15microM 4.0s(-1), and 0.27microM(-1)s(-1)). Fluoride potently inhibits diadenosine tetraphosphate hydrolysis in the presence of Mg(2+) (IC(50)=20microM), whereas it is ineffective in the presence of Zn(2+), supporting the view that inhibition involves a specific, MgF(3)(-)-containing transition state analogue complex. Patterns of Apf expression in Drosophila tissues show Apf mRNA levels to be highest in embryos and adult females. Subcellular localization with Apf-EGFP fusion constructs reveals Apf to be predominantly nuclear, having an apparent preferential association with euchromatin and facultative heterochromatin. This supports a nuclear function for diadenosine tetraphosphate. Our results show Apf to be a fairly typical member of the bis (5'-nucleosyl)-tetraphosphatase subfamily of Nudix hydrolases with features that distinguish it from a previously reported bis (5'-nucleosyl)-tetraphosphatase hydrolase activity from Drosophila embryos.
Winward
Lucinda
L
Division of Cell Signalling, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. lusi83@hotmail.com
Whitfield
William G F
WG
Woodman
Timothy J
TJ
McLennan
Alexander G
AG
Safrany
Stephen T
ST
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D015032:Zinc / Q000494:pharmacology; D000096964:Nudix Hydrolases
D000255:Adenosine Triphosphate; D015226:Dinucleoside Phosphates; D029721:Drosophila Proteins; D049452:Green Fluorescent Proteins; D008274:Magnesium; D011755:Pyrophosphatases; D011993:Recombinant Fusion Proteins; D015032:Zinc; D000096964:Nudix Hydrolases; C070070:diadenosine 5',5''''-P1,P6-hexaphosphate