Isolation of serine acetyltransferase complexed with cysteine synthase from Allium tuberosum.

Abstract

Serine acetyltransferase (SATase, EC 2.3.1.30), an enzyme which catalyzes the biosynthesis of O-acetyl-L-serine (OAS) from L-serine and acetyl-CoA, has been purified about 33, 000-fold with a yield of 11 % from leaf extracts of Allium tuberosum with a procedure involving heat treatment, ammonium sulfate fractionation, DEAE-Toyopearl chromatography, Ultrogel AcA34 gel filtration, and Blue-Toyopearl affinity chromatography, followed by a second filtration through Ultrogel AcA34. The isolated enzyme was apparently homogeneous as shown by PAGE with a specific activity of 231 units/mg protein. Besides this SATase activity the enzyme also had a significant level of cysteine synthase (CSase, EC 4.2.99.8) activity with a specific activity of 39.2 units/mg protein. The molecular weight of the enzyme was 650, 000 by gel filtration. Subunit analysis by SDS-PAGE yielded two kinds of protein bands with a large subunit molecular weight of 35, 000 and a small subunit molecular weight of 31, 000. The results of Western blotting analysis using anti-rape CSase IgG suggested that the large subunk could be the CSase subunit.