Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum.

Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases. One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite. The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa. Among the triphosphates of various nucleosides tested, ATP was the best substrate. The enzyme exhibited a Km of 0.5 mM for ATP and a Vmax of 109 micromol ATP (mg protein)(-1) min(-1); the optimum pH for activity was near 6.5. The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide. Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.