The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera.
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.
Siebers
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Universität Osnabrück, Federal Republic of Germany.
Altendorf
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D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D000917:Antibody Formation; D000918:Antibody Specificity; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005838:Genotype; D007106:Immune Sera / Q000302:isolation & purification*; D015151:Immunoblotting; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D011489:Protein Denaturation; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D013379:Substrate Specificity
D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D007106:Immune Sera; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium