In Vivo Mitochondrial Pyruvate Dehydrogenase Activity, Studied by Rapid Fractionation of Barley Leaf Protoplasts

Abstract

Inactivation and activation of mitochondrial and chloroplastic pyruvate dehydrogenase (PDH; EC 1.2.4.1) have been studied in isolated organelles and protoplasts from barley leaves. The pyruvate dehydrogenase complex (PDC) from barley leaf mitochondria was inactivated by ATP (65% at 4 mM ATP) while the chloroplastic PDC was stimulated (75% at 4 mM ATP). MgCl2 inhibited the mitochondrial complex with increasing concentrations while the chloroplastic complex was stimulated. MnCl2 had a stimulatory effect on both PDCs. ATP-inactivated mitochondrial PDC could be reactivated by MnCl2, but not by MgCl2 or CaCl2. The major part of the PDC was located in the chloroplast. The in vivo mitochondrial PDC activity could be determined after removal of the chloroplastic isoform by subcellular fractionation. This activity was sensitive to ATP inhibition confirming the mitochondrial origin. The in vivo PDC activity as well as the ATP sensitivity did not change when protoplasts were incubated in darkness or illuminated in photorespiratory or non-photorespiratory conditions. This was taken as an indication of an unchanged activation state of the enzyme under the conditions tested.