Characterization of a soluble inorganic pyrophosphatase from Microcystis aeruginosa and preparation of its antibody.
A soluble inorganic pyrophosphatase was isolated from a crude extract of Microcystis aeruginosa by adsorption chromatography. The enzyme was purified to homogeneity as judged by sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide gel electrophoresis and N-terminal amino acid analysis. The molecular mass was estimated to be 80 kDa by gel filtration chromatography, 87 kDa by nondenaturing polyacrylamide gel electrophoresis, and 28 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an isoelectric point of 4.5, which is similar to the pI values reported for other soluble inorganic pyrophosphatases. The sequence of 29 N-terminal amino acids was determined; only 4 of these amino acids are identical to those in the sequence of Saccharomyces cerevisiae inorganic pyrophosphatase. M. aeruginosa inorganic pyrophosphatase is a Mg(2+)-dependent enzyme exhibiting a pH optimum of around 7.5. Its KM value for inorganic pyrophosphate was estimated to be 1.30 mM. A specific antibody was raised in chicken to M. aeruginosa inorganic pyrophosphatase. No immunological cross-reactivity was seen when Western blots of partially purified S. cerevisiae or Escherichia coli inorganic pyrophosphatase were probed with the antibody.
Kang
C B
CB
Botany Department, National University of Singapore.
Ho
K K
KK
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D046931:Microcystis / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D000906:Antibodies; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase