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-mu-mol 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.