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

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 electrophoresis. 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+ concentrations increase. The change in threonine and K+ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonineinsensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.

Immunological Characterization of in Vitro Forms of Homoserine Dehydrogenase from Carrot Suspension Cultures

“…HSDH II has a mol wt range of 150,000 to 190,000. HSDH III has a mol wt range of 300,000 to 400,000, and HSDH IV has mol wt greater than 400 (23). However, these conversions were observed at potassium concentrations of 250 mm, which are approximately 2 to 2.5 times higher than the potassium concentrations that have been observed for isolated pea (3, 1 1) and spinach chloroplast (8,26).…”

“…It is composed of four subunits having mol wt of 86,000 ± 4000. AK-II/HSDH-II has a mol wt of 185,000, and it is composed of two identical subunits of 88,000 (6 Carrot HSDH is composed of identical 85,000 Mr subunits, the T-form appears to be a homotrimer and the K-form appears to be a homodimer (23). The antiserum was tested for its ability to detect the 85,000 Mr subunits.…”

“…HSDH has been localized in the chloroplast fractions of maize (4) and pea leaves (24,25). To date, HSDH has been purified to homogeneity and well characterized from maize (18) and recently in our laboratory from carrot suspension cultures (23 …”

“…Vol. 92,1990 would then favor the formation of the T-form, which is threonine sensitive and is less responsive to stimulation by potassium ions than the K-form (23). Under these conditions, HSDH activity and threonine biosynthesis would be curtailed.…”

Bifunctional Protein in Carrot Contains Both Aspartokinase and Homoserine Dehydrogenase Activities

“…AK activity has been separated into two forms that are inhibited by either lysine or threonine. HSDH has been purified to apparent homogeneity and characterized (16 …”