Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase.
The ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are a family of enzymes that control the levels of extracellular nucleotides, thereby modulating purinergically controlled physiological processes. Six of the eight known NTPDases are membrane-bound enzymes; only NTPDase 5 and 6 can be released as soluble enzymes. Here we report the first bacterial expression and refolding of soluble human NTPDase5 from inclusion bodies. The results show that NTPDase5 requires the presence of divalent cations (Mg2+ or Ca2+) for activity. Positive cooperativity with respect to hydrolysis of its preferred substrates (GDP, IDP and UDP) is observed, and this positive cooperativity is attenuated in the presence of nucleoside monophosphate products (e.g., GMP and AMP). In addition, comparing the biochemical properties of wild-type NTPDase5 and those of a mutant NTPDase5 (C15S, which lacks the single, non-conserved cysteine residue), also expressed in bacteria, suggests that Cys15 is not essential for either proper refolding or enzymatic activity (indicating this residue is not involved in a disulfide bond). Moreover, the substrate profile of bacterially expressed NTPDase5, as well as the C15S mutant, was determined to be similar to that of full-length, membrane-bound and soluble NTPDase5 expressed in mammalian COS cells.
Murphy-Piedmonte
Deirdre M
DM
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, P.O. Box 670575, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, USA.
Crawford
Patrick A
PA
Kirley
Terence L
TL
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D015964:Gene Expression Regulation, Bacterial; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D015513:Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D011755:Pyrophosphatases; D012995:Solubility; D013379:Substrate Specificity
D002413:Cations, Divalent; D004220:Disulfides; D015513:Oncogene Proteins; D006153:Guanosine Diphosphate; D000251:Adenosine Triphosphatases; C117634:ENTPD5 protein, human; D011755:Pyrophosphatases; C027201:ectoATPase; D008274:Magnesium; D002118:Calcium