The K+‐translocating Kdp‐ATPase from Escherichia coli

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Abstract
The Kdp system from Escherichia coli is a derepressible high-affinity K+ -uptake ATPase. Its membrane-bound ATPase activity was approximately 50 μmol g−1 min−1. The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 μM) and Mg2+-ATP (Km= 80 μM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki= 1.5 μM), fluorescein isothiocyanate (Ki= 3.5 μM), N,N′-dicyclohexylcarbodiimide (Ki= 60 μM) and N-ethylmaleimide (Ki= 0.1 mM). The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate. Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS. Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme. In contrast, the anti-KdpC serum did not inhibit enzyme activity.