Geisler M, Koenen W, Richter J, Schumann J
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Germany.
Biochim Biophys Acta. 1998 Jan 19;1368(2):267-75. doi: 10.1016/s0005-2736(97)00193-4.
In a previous paper, we published the sequence of a P-type ATPase gene from Synechocystis 6803 [Geisler et al. (1993) J. Mol. Biol. 234, 1284] which showed significant homologies to eukaryotic calcium ATPases. To investigate the specificity and activities of this plasma membrane-bound enzyme, we expressed the slightly modified gene in an ATPase deficient E. coli strain. The expressed ATPase showed an apparent molecular mass of about 97kDa and is localized in the E. coli plasma membranes. The introduced 6xHis tag at the N-terminus allowed the purification of the Synechocystis 6xHis-ATPase by single-step affinity chromatography using a Ni2+-nitrilotriacetic acid resin. The ATPase activity of the enzyme is inhibited by vanadate (IC50 = 119 microM), N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, and inhibitors of eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases; however, it is stimulated by thapsigargin. Formation of phosphorylated enzyme intermediates depends on calcium ions indicating that the Synechocystis P-ATPase acts as a calcium pump equivalent to eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases.
