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- Title
Purification and Characterization of Homo- and Hetero-Dimeric Acetate Kinases from the Sulfate-Reducing Bacterium Desulfovibrio vulgaris1.
- Authors
Yu, Ling; Ishida, Tetsuo; Ozawa, Kiyoshi; Akutsu, Hideo; Horiike, Kihachiro
- Abstract
Two distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (α2s) and the other (AK-II) was a heterodimer (αSαL). On SDS-PAGE, αL and αS subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg2+ requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.
- Subjects
DESULFOVIBRIO vulgaris; PROTEINS; PROTEOMICS; BIOSYNTHESIS; AMINO acids
- Publication
Journal of Biochemistry, 2001, Vol 129, Issue 3, p411
- ISSN
0021-924X
- Publication type
Article