Characterization of Rat NTPDase1, -2, and -3 ectodomains refolded from bacterial inclusion bodies.
The ecto-nucleoside triphosphate diphosphohydrolases or NTPDases are a family of membrane-bound enzymes that catalyze the sequential removal of gamma- and beta-phosphate from ATP, ADP, and other nucleotides. NTPDase1, -2, -3, and -8 are the enzymes responsible for signal conversion and termination in purinergic signaling. They are anchored to the cytoplasmic membrane by two transmembrane helices with a large catalytic domain pointing toward the extracellular space. Here we report the first successful expression and purification of the soluble extracellular domains of rat NTPDase1, -2, and -3 from bacterial inclusion bodies. The refolded proteins show characteristics similar to the wild type enzymes, for example in that they are dependent on divalent metal ions for catalysis and hydrolyze a wide variety of nucleoside tri- and diphosphates, whereas the monophosphate AMP is not further degraded. Nucleoside triphosphates are hydrolyzed at a higher rate than the corresponding diphosphates. Other characteristics of the recombinant enzymes however reflect the absence of transmembrane regions and side chain glycosylation. For example all three enzymes are monomeric and only subtly activated by Mg2+ ions as compared to Ca2+ ions. Although having a considerably higher specificity constant kcat/Km for ADP as for ATP, the bacterially expressed variant of NTPDase1 in contrast to its wild type counterpart releases intermediate ADP to a substantial amount. The presented expression system will allow large scale production of active protein suitable for structural studies, development of inhibitors, and even clinical application.
Zebisch
Matthias
M
Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Sträter
Norbert
N
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D015703:Antigens, CD; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; C027201:ectoATPase; C439652:nucleoside-triphosphate diphosphohydrolase 3; D001081:Apyrase; C067869:CD39 antigen