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

The enzyme system catalysing the conversion of D-glucuronolactone and L-gulonolactone into Lascorbic acid in animal tissues has been previously reported to be entirely located in the microsomal fractions ofratand goat-liver homogenates (Burns, Peyser & Moltz, 1956; Chatterjee, Ghosh, Ghosh & Guha, 1957a, b; 1958a, b). The kidney tissue has been found to be the site of this conversion in amphibian, reptilian and some of the avian species examined (Roy & Guha, 1958), and in chick kidney the enzyme system concerned has also been found to be located in the microsomes. Isherwood (1953) stated that mitochondria from rat liver catalyse the transformation of L-gulonolactone into L-ascorbic acid. However, our experiments indicate that the observed activity of mitochondria is probably due to their contamination by microsomes. Potassium cyanide greatly accelerates this synthesis from Dglucuronolactone but the synthesis from L-gulonolactone does not require cyanide or any other added factor (Chatterjee et al. 1958 b). Subsequently the conversion of L-gulonolactone into L-ascorbic acid by rat-liver microsomes has been found to be greatly accelerated by boiled rat-liver supernatant or a metal-binding agent such as sodium pyrophosphate, oco'-dipyridal and 8-hydroxyquinoline (Chatterjee, Chatterjee, Ghosh, Ghosh & Guha, 1958c). The microsomal enzyme concerned in the oxidation of L-gulonolactone to L-ascorbic acid is inhibited by heavy-metal ions (Hg2+, Cu2+ and Zn2+) and reversibly by p-chloromercuribenzoate, indicating the involvement of some essential thiol groups in the enzyme (Chatterjee et al. 1958c). It has already been reported that the microsomes can convert only the lactone forms of the precursors, namely D-glucuronolactone and Lgulonolactone. The sodium salts of the corresponding free acids are not acted on (Chatterjee et al. 1957b; 1958 a, b). The soluble supernatant has been found to inhibit the rate of conversion of L-gulQnolactone into L-ascorbic acid to a great extent and, though L-gulonate is not converted by the microsomes alone, it leads to the formation of L-ascorbic acid when soluble supernatant is added to the system. The factor in the supernatant which inhibits the rate of microsomal synthesis of ascorbic acid from L-gulonolactone but, curiously, helps in