Subcellular localization of hexokinase in pea leaves. Evidence for the predominance of a mitochondrially bound form.

Hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) activity was determined in subcellular fractions prepared from pea (Pisum sativum) leaf homogenates. About 60% of the total detectable activity of hexokinase was found associated with a particulate fraction consisting essentially of mitochondria and chloroplasts and free of cytosol contamination. The hexokinase specific activity of the particulate fraction was 2-fold higher than that of the homogenate and about 4-fold higher than that of the cytosol. Using a specially designed isokinetic-isopycnic sucrose density gradient centrifugation method, the distribution of hexokinase activity correlated with that of the mitochondrial marker (cytochrome oxidase) and not with that of the chloroplast membrane marker ( chlorophyll ) or that of the cytosol marker (phosphoenolpyruvate carboxylase). Thus, the hexokinase/mitochondria ratio was close to 1.0 along the entire gradient, while the hexokinase/chloroplast ratio varied over a 10-fold range. The results strongly suggest that hexokinase is predominantly bound to mitochondria of pea leaves, and that pea leaf chloroplasts are essentially devoid of any specifically associated hexokinase activity. This work provides the first demonstration of a specific association of hexokinase with mitochondria from photosynthetic tissues of higher plants.