PMID:2849541
| Citation |
Siebers, A and Altendorf, K (1988) The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera. Eur. J. Biochem. 178:131-40 |
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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 mumol 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 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) 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. |
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| Keywords |
Adenosine Triphosphatases/antagonists & inhibitors; Adenosine Triphosphatases/immunology; Adenosine Triphosphatases/isolation & purification; Antibody Formation; Antibody Specificity; Cation Transport Proteins; Cell Membrane/enzymology; Chromatography, Gel; Chromatography, Ion Exchange; Cytoplasm/enzymology; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Genotype; Immune Sera/isolation & purification; Immunoblotting; Mutation; Potassium/metabolism; Protein Denaturation; Sodium-Potassium-Exchanging ATPase/genetics; Sodium-Potassium-Exchanging ATPase/immunology; Sodium-Potassium-Exchanging ATPase/isolation & purification; Substrate Specificity |
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