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

Abstract

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