Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura
1996
Hohlfeld, H. | Scheel, D. | Strack, D.
A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1] which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-COA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat.1kg protein(-1). Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-COA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca2+. The apparent Km value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the Km value for tyramine was about tenfold greater (174 micromolar) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 micromolar).
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