Publications

• The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera.

  Siebers A., Altendorf K.

  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-S ... >> More

  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. << Less

  Eur. J. Biochem. 178:131-140(1988) [PubMed] [EuropePMC]

• Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli.

  Gassel M., Siebers A., Epstein W., Altendorf K.

  Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF. The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them i ... >> More

  Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF. The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them into the cytoplasmic membrane. In the strains that do not express KdpC or KdpA the other two subunits did not copurify on dye-ligand affinity columns after solubilization with non-ionic detergent. In the mutant lacking KdpB the other two subunits copurified under the same conditions. It is concluded that KdpC forms strong interactions with the KdpA subunit, serving to assemble and stabilise the Kdp complex. A structure in which KdpC could be one of the connecting links between the energy-delivering subunit KdpB and the K+-transporting subunit KdpA is suggested by these data. << Less

  Biochim. Biophys. Acta 1415:77-84(1998) [PubMed] [EuropePMC]

• Physicians, ethics, advance directives, and long-term care.

  Gordon M., Levitt D.

  J Am Geriatr Soc 44:471-472(1996) [PubMed] [EuropePMC]

• Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli.

  Siebers A., Altendorf K.

  During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on t ... >> More

  During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP. << Less

  J Biol Chem 264:5831-5838(1989) [PubMed] [EuropePMC]