Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures

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Abstract
Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electronphoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 .+-. 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K+. When the enzyme is dialyzed in the presence of K+ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K+. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K+ concentration increase. The change in threonine and K+ concentrations shift the homoserine dehydrogenase from threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.