Intracellular Localization of Aspartate Kinase and the Enzymes of Threonine and Methionine Biosynthesis in Green Leaves

The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine γ-synthase are confined to the chloroplast. Cystathionine β-lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm

Distribution of the Enzymes of Nitrogen Assimilation within the Pea Leaf Cell

Protoplasts obtained from expanded leaves of Pisum sativum have been used for the isolation of cell organelles and the subsequent study of the intracellular distribution of the enzymes of nitrate assimilation. The protoplasts were ruptured in a suitable medium and the total lysate subjected to sucrose density gradient centrifugation. Of the total chlorophyll more than 80% was recovered in intact chloroplasts. Nitrite reductase and glutamate synthase were found to be located wholly in the chloroplast. Glutamine synthetase was distributed between the chloroplast and the cytoplasm, with a maximum of 60% of the former. A possible role of the cytoplasmic enzyme is discussed in relation to photorespiration. There was no evidence for the association of nitrate reductase with any cell organelle or membrane fraction