Biological synthesis of l-ascorbic acid in animal tissues: conversion of l-gulonolactone into l-ascorbic acid

Abstract

The enzyme catalyzing the conversion of L-gulonolactone into L-ascorbic acid was found in the liver microsomes of the rat and the goat and kidney microsomes of the chick. The synthesis of ascorbic acid from gulonolactone by rat-liver and chick-kidney microsomes was accelerated by the metal-binding agents sodium pyrophosphate, act [alpha][alpha][image]-dipyridyl and 8-hydroxyquinoline and by the boiled supernatant from livers of rat, goat and guinea pig, and kidney of the chick. These factors did not accelerate the synthesis of ascorbic acid by goat-liver microsomes. The enzyme converting L-gulonolactone into L-ascorbic acid was inhibited by heavy-metal ions (Hg2+, Cu2+, Zn2+) and by p-chloromercuribenzoate. The inhibition by the last-named was reversible with reduced glutathione, indicating the involvement of thiol groups in the enzyme. The conversion of L-gulonolactone into L-ascorbic acid by a soluble and purified preparation of the microsomal enzyme was inhibited by the flavin inhibitors riboflavin, Antabuse (disulfiram) and riboflavin monosulphate, which indicated the involvement of a flavo-protein. With sodium L-gulonate as substrate the soluble enzyme preparation did not reduce diphosphopyridine nucleotide and no xylulose formation was detected. L-Gulonolactone and not L-gulonate was the precursor of ascorbic acid. Gulonate was converted into ascorbic acid only after lactonization by the action of gulonolactonase, an enzyme present in the soluble supernatant. The action was reversible and the equilibrium favored hydrolysis of the lactone. A scheme showing the pathways of the biosyntheses of L-ascorbic acid and of L-xylulose was given. The conversion of L-gulonolactone into L-ascorbic acid was shown to follow a different and independent pathway from that of L-gulonolactone into L-xylulose. The former was catalyzed by an enzyme present entirely in the microsomes and the latter by an enzyme system present only in the soluble supernatant. The possibility of L-xylohexulonolactone (2-keto-L-gulonolactone) being an intermediate in the synthesis of L-ascorbic acid was discussed. In guinea-pig liver, the microsomal enzyme catalyzing the conversion of L-gulonolactone into L-ascorbic acid was missing.