Purification and characterization of the Ca2+-ATPase of Flavobacterium odoratum.
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.
Desrosiers
M G
MG
Cardiology Division, Department of Medicine, Medical University of South Carolina, Charleston, 29425-2221, USA.
Gately
L J
LJ
Gambel
A M
AM
Menick
D R
DR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D014638:Vanadates / Q000494:pharmacology
D010446:Peptide Fragments; D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium