EXPRESSION AND CHARACTERIZATION OF A SYNECHOCYSTIS PCC-6803 P-TYPE ATPASE IN ESCHERICHIA-COLI PLASMA-MEMBRANES

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, 128 4] 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 def icient E. coli strain. The expressed ATPase showed an apparent molecul ar mass of about 97 kDa and is localized in the E. coli plasma membran es. The introduced 6xHis tag at the N-terminus allowed the purificatio n of the Synechocystis 6xHis-ATPase by single-step affinity chromatogr aphy using a Ni2+-nitrilotriacetic acid resin. The ATPase activity of the enzyme is inhibited by vanadate (IC50 = 119 mu M), N-ethylmaleimid e, N,N-dicyclohexylcarbodiimide, and inhibitors of eukaryotic sarco(en do)plasmic reticulum Ca2+-ATPases; however, it is stimulated by thapsi gargin. Formation of phosphorylated enzyme intermediates depends on ca lcium ions indicating that the Synechocystis P-ATPase acts as a calciu m pump equivalent to eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPa ses. (C) 1998 Elsevier Science B.V.