Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus.
The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.-) ( DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having Mr values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (pI = 4.7-5.3) plus two minor protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1 pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.[1]References
- Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus. Power, R., Kurz, W.G., De Luca, V. Arch. Biochem. Biophys. (1990) [Pubmed]
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