Purification and Characterization of the Ca-ATPase of Flavobacterium odoratum

The P-type Ca -ATPase from Flavobacterium odoratum has been purified to homogeneity and characterized. Inside-out membrane vesicles were extracted with C E , followed by ammonium sulfate fractionation, centrifugation through two successive 32-48% glycerol gradients, and DE52 ion exchange chromatography. The purified Ca -ATPase consists of a single polypeptide. It migrates electrophoretically with an apparent molecular mass of 60,000 Da, consistent with the phosphorylation pattern originally reported in membrane vesicles. This single polypeptide is functional and capable of calcium-dependent vanadate-sensitive ATP hydrolysis and of forward and reverse phosphorylation. Maximum hydrolysis activity occurs at pH 8.0, with a specific activity of 75 μmol of ATP hydrolyzed min mg protein. The purified Ca -ATPase has an apparent K for calcium of 1.5 μM and for ATP of 90 μM. Vanadate strongly inhibits the activity with an IC of 0.6 μM. The prokaryotic Ca -ATPase is insensitive to the SR Ca -ATPase inhibitors fluorescein isothiocyanate, thapsigargin, and cyclopiazonic acid. It is rapidly phosphorylated by [ - P]ATP in a calcium-dependent vanadate-inhibited manner and can be phosphorylated by P in both the presence and absence of calcium.