Biosynthesis of ascorbic acid in kidney bean. L-galactono-gamma-lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane

Hypocotyls of kidney beans (Phaseolus vulgaris L.) accumulated ascorbate af ter preincubation with a number of possible precursors, mainly L-galactono- gamma-lactone (L-GL) and L-gulono-gamma-lactone. The increase in the intrac ellular ascorbate concentration was parallel to the high stimulation of the L-GL dehydrogenase (L-GLD) activity measured in vitro using L-GL as a subs trate and cytochrome c as an electron acceptor. Cell fractionation using a continuous linear Percoll gradient demonstrated that L-GLD is associated wi th mitochondria; therefore, pure mitochondria were isolated and subjected t o detergent treatment to separate soluble from membrane-linked proteins. L- GLD activity was mainly associated with the detergent phase, suggesting tha t a membrane-intrinsic protein is responsible for the ascorbic acid biosynt hetic activity. Subfractionation of mitochondria demonstrated that L-GLD is located at the inner membrane.