The pathway of lysine degradation via aminoadipic acid has been investigated in maize endosperm. The first two enzymes, lysine-ketoglutarate reductase and saccharopine dehydrogenase, catalyze the following reactions:
The enzymes were extracted from a number of inbreds and hybrids, according to the procedure of Arruda et al. (Plant Physiol. 69:988-989,1982). The endosperms were isolated from immature seeds and ground in a chilled mortar with 100 mM potassium phosphate buffer, pH 7.0, containing 1mM EDTA and 1 mM 2-mercaptoethanol. The homogenate was centrifuged at 12,000g for 25 min and the supernatant treated with (NH4)2SO4 to 70% saturation. The precipitate obtained was collected and taken up in 3 ml of extraction buffer. The extract was desalted on a Sephadex G25 column (1 x 9 cm) and the protein fraction was assayed for lysine-ketoglutarate reductase and saccharopine dehydrogenase. For LKR the assay mixture contained potassium phosphate pH 7.0 (100 umol), L-lysine (18 umol), alpha-ketoglutarate (9 umol), NADPH (100 umol) and various concentrations of protein in a total volume of 1 ml. For saccharopine dehydrogenase the assay mixture consisted of saccharopine (1 umol), NAD (1 umol), Tris-HCl pH 8.6 (100 umol) and various concentrations of protein.
For both enzymes the activity was linear with time and proportional to the amount of extract added to the assay. The apparent Km values of LKR for lysine and alpha-ketoglutarate were 5.2 and 1.8 mM respectively (Table 1). For SDH the Km values for saccharopine and NAD were 0.05 and 0.2 respectively (Table 1). The enzymes show differences with respect to cofactor and pH. LKR is specific for NADPH and has a pH optimum close to 7.0. SDH utilizes NAD as cofactor and shows a pH optimum close to 8.6.
The activity of LKR and SDH of some hybrids is shown in Table 2. LKR activity increases with the onset of seed development, reaches a peak at middle stage, and decreases at seed maturity. Saccharopine dehydrogenase was analyzed only at 30 days after pollination, when it showed an activity of 7.2 nmol of NAD reduced per endosperm per min.
We investigated the activity of LKR and SDH in some opaque-2 strains. For this purpose we used two inbred lines, ML649 and L438 and a tropical maize variety named Maya, each containing homozygous normal and opaque-2 strains. The LKR activity of L438 opaque-2 endosperm was three times less than the activity observed for L438 normal. In the ML649 inbred line, enzyme activity of opaque-2 endosperm was practically absent as compared with normal endosperm (Table 3). The decrease in LKR activity of opaque-2 endosperm cannot be attributed to the presence of an enzyme inhibitor in the mutant endosperm. Assays conducted with mixtures of enzyme extracts from normal and opaque-2 endosperms showed an activity equal to the sum of the individual activities.
The activity of SDH was not changed by the opaque-2 gene. The activity of this enzyme for normal and opaque-2 versions of the Maya variety was basically the same (Table 3).
The results found here show for the first time that the degradation of lysine is under the control of the opaque-2 gene. Opaque-2, by decreasing the activity of LKR, probably decreases the rate of lysine degradation in the endosperm. This is in good agreement with the decreased extent of lysine breakdown observed in the opaque-2 endosperm by Sodek and Wilson (Arch. Biochem. Biophys., 140:29-36, 1970). The opaque-2 gene, however, affects only the first enzyme in the lysine degradation pathway but does not affect the second enzyme. This finding points out that lysine degradation is an important means by which opaque-2 increases lysine content in the endosperm.
P. Arruda, J. M. G. Tocozzilli, A. Vieira and W. J. da Silva
Return to the MNL 58 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page