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.
The 60-kDa Ca2+-ATPase from Flavobacterium odoratum is kinetically and mechanistically similar to other P-type ATPases, suggesting its use as a model system for structure-function studies of ion transport. A portion of the gene was amplified by polymerase chain reaction of genomic DNA with degenerate oligonucleotide primers, one based on the N-terminal amino acid sequence of the purified protein and the other based on a consensus sequence for the phosphorylation site of P-type ATPases. This gene fragment was used to screen a lambda library of F. odoratum 29979 DNA. Clone "C" is 3.3 kilobases in length and contains one complete and part of a second open reading frame, the first of which encodes a 58-kDa protein containing the exact N-terminal amino acid sequence of the purified protein. We have named this gene cda, for calcium-dependent ATPase. Escherichia coli, transformed with clone C, demonstrates high levels of calcium-dependent and vanadate-sensitive ATP hydrolysis activity, forms a 60-kDa phosphointermediate, and cross-reacts with antibodies to the purified Ca2+-ATPase. The gene has almost no sequence homology to even the highly conserved regions characteristic of P-type ATPases but does possess significant homology to a protein with alkaline phosphatase activity (PhoD) from Zymomonas mobilis. The putative phosphorylation site is a Walker A (P-loop) ATP binding sequence and is modified relative to P-type ATPases, suggesting that the F. odoratum Ca2+-ATPase may represent an ancestral link between the F- and the P-type ATPases or perhaps a new class of ATPases.
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