Purification and Characterization of the Ca2+-ATPase of Flavobacterium odoratum

Abstract

The P-type Ca2+-ATPase from Flavobacterium odoratum has been purified to homogeneity and characterized. Inside-out membrane vesicles were extracted with C12E8, followed by ammonium sulfate fractionation, centrifugation through two successive 32-48% glycerol gradients, and DE52 ion exchange chromatography. The purified Ca2+-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 approximately 75 micromol of ATP hydrolyzed min-1 mg-1 protein. The purified Ca2+-ATPase has an apparent Km for calcium of 1.5 microM and for ATP of 90 microM. Vanadate strongly inhibits the activity with an IC50 of 0.6 microM. The prokaryotic Ca2+-ATPase is insensitive to the SR Ca2+-ATPase inhibitors fluorescein isothiocyanate, thapsigargin, and cyclopiazonic acid. It is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent vanadate-inhibited manner and can be phosphorylated by Pi in both the presence and absence of calcium.

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes

“…Collet, V. Stroobant, and E. van Schaftingen, submitted for publication). The PSI‐BLAST and HMMmer searches allowed us to align the corresponding regions in other enzymes of the AlkP superfamily, such as alkaline phosphodiesterase/nucleotide pyrophosphatase (autotaxin) PC‐1,21, 22 streptomycin‐6‐phosphatase,23 Ca 2+ ‐dependent ATPase,24 phosphonoacetate hydrolase,25 phosphonate monoesterase,26 and GPI‐anchoring enzymes (glycosylphosphatidylinositol phosphoethanolaminetransferases) MCD4, GPI7, and GPI1327–30 (Fig. 2).…”

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

“…As pointed out by Rosen (1987), the bacterial Ca 2+ pump is located near the outside of the cell, whereas the Mg 2+ pump is located towards the inside. Normal intracellular calcium concentrations are usually up to 103 times lower than extracellular concentrations (Desrosiers et al 1996;Norris et al 1996). These observations could explain the greater tendency of these bacterial strains to precipitate calcium carbonates.…”

The Chryseobacterium meningosepticum PafA enzyme: prototype of a new enzyme family of prokaryotic phosphate-irrepressible alkaline phosphatases? The GenBank accession number for pafA reported in this paper is AF157621.

“…PhoD of Z. mobilis and the Ca# + -ATPase of M. odoratus also demonstrated the highest activity on ATP (Gomez & Ingram, 1995 ;Desrosiers et al, 1996). However, Cda is a narrowspectrum enzyme as it is able to hydrolyse essentially only ATP , whilst PhoD is active on different substrates (Gomez & Ingram, 1995).…”