title: Rapid, high yield purification and characterization of the K(+)-translocating Kdp-ATPase from Escherichia coli.
abstract: The conventional procedure for the purification of the high affinity K+ uptake ATPase (KdpABC) from Escherichia coli involves a tedious three-column protocol (final enzyme purity, approximately 90%; activity yield, 6.5% (Siebers, A., and Altendorf, K. (1988) Eur. J. Biochem. 178, 131-140)). We have now developed a highly effective one-column (Fractogel TSK AF-Red) protocol yielding an enzyme preparation of comparable purity with severalfold higher activity yield. A further increase in enzyme purity up to 98% was achieved by a two-column protocol involving elution over DEAE-Sepharose CL-6B prior to TSK AF-Red affinity chromatography. The reduction of preparation time minimized KdpB protein degradation and led to hitherto unequaled values of specific activity (up to 2000 mumols x g-1 x min-1) and enrichment factors (up to 30-fold). Our results confirm the usefulness of triazine dye matrices for the purification of transport ATPases.
authors.affiliation: Universität Osnabrück, Arbeitsgruppe Mikrobiologie, Federal Republic of Germany.
authors.forename: A
authors.initials: A
authors.lastname: Siebers
authors.forename: R
authors.initials: R
authors.lastname: Kollmann
authors.forename: G
authors.initials: G
authors.lastname: Dirkes
authors.forename: K
authors.initials: K
authors.lastname: Altendorf
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D027682:Cation Transport Proteins; D002846:Chromatography, Affinity; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D011108:Polymers; D014227:Triazines
chemical_list: D027682:Cation Transport Proteins; D004396:Coloring Agents; D029968:Escherichia coli Proteins; D011108:Polymers; D014227:Triazines; C075445:Fractogel TSK AF-Red; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli