[Abstract]  [Full Text]  [PDF]

Markus Geisler^1*

¹ Institut für Biochemie der Pflanzen. Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf. Germany.

* To whom correspondence should be addressed: Markus Geisler, Institut für Biochemie der Pflanzen. The Royal Veterinary and Agricultural University, Department of Plant Biology, Plant Physiology/Anatomy Laboratory, Thorvaldsensvej 40, DK-1871 Frederiksberg C. Denmark. Email: mag@staff.kvl.dk

Biol. Proced. Online 1998;1:70-80. doi:10.1251/bpo9
Submitted: March 20, 1998; Published: May 14, 1998.

Indexing terms: Ca(2+)-Transporting ATPase; Escherichia coli; chromatography.

Abbreviations: EP, phosphoenzyme; IPTG, isopropyl-β-D-thiogalactopyrano-side; Ni-NTA, Ni²⁺-nitrilotriacetic acid; PM, plasma membrane; PMCA, plasma membrane Ca²⁺-ATPase(s); SER, sarco(endo)plasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca²⁺-ATPase(s); 6xHis, 6 x histidine affinity tag.

In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993) et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC₅₀= 119 μM) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.