Ascorbate biosynthesis in mitochondria is linked to the electron transportchain between complexes III and IV

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tis sues. An improved extraction procedure involving ammonium sulfate precipita tion of membrane proteins from crude leaf homogenates yielded a simple, qui ck method for determining tissue activities of galactono-gamma-lactone dehy drogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with l eaf age. Subcellular fractionation experiments using marker enzymes demonst rated that 80% of the total GLDH activity was located on the inner mitochon drial membrane, and 20% in the microsomal fraction. Specific antibody raise d against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polyp eptide in extracts from the mitochondrial membranes but failed to detect th e equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mit ochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isola ted intact mitochondria. This evidence suggests that GLDH delivers electron s to the mitochondrial electron transport chain between complexes III and I V.