Isolation, chacterization and regulation of isoenzymes of aspartate kinase differentially sensitive to calmodulin from spinach leaves

Abstract

Two isofunctional forms of aspartate kinase, one sensitive to lysine (isoenzyme I) and the other to threonine (isoenzyme II) have been isolated from spinach leaves by DEAE- and Sephadex G-200 column chromatography with molecular weights of 330 000 and 200 000, respectively. These forms differ also in their sensitivity to Ca²⁺ and calmodulin. While the form inhibited by lysine is insensitive to Ca²⁺ and calmodulin, the form that is inhibited by threonine is activated 3-4-fold by calmodulin. The effect fo calmodulin is furtehr increased by Ca²⁺ and is inhibited by EGTA, which specifically chelates out Ca²⁺. This inhibition can be relieved by Ca²⁺. The apparent K_m values for asparatate and ATP in the case of the lysine-sensitive isoenzyme, isoenzyme I, which is unaffected by calmodulin, also remain unaffected by calmodulin. However, the apparent K_m values for the two substrates are reduced in the presence of calmodulin in the case of the threonine-sensitive isoenzyme, isoenzyme II.