Effect of cyanide on the biosynthesis of ascorbic acid by an enzyme preparation from goat-liver tissue.

In exploring the mechanism of biosynthesis of ascorbic acid by the rat, King and his coworkers (Musulin, Tully, Longenecker & King, 1939; Longenecker, Musulin, Tully & King, 1939; Longenecker, Fricke & King, 1940) observed that rats treated with Chloretone (1:1:1-trichloro-2methylpropan-2-ol) excreted more ascorbic acid than did the controls. By employing Chloretonized rats and isotopically labelled precursors they (Jackel, Mosbach, Burns & King, 1950; Horowitz, Doerschuk & King, 1952; Horowitz & King, 1953a, b) presented evidence for the following route of the synthesis: D-Glucose -+ D-glucurono-y-lactone -* L-ascorbic acid. Subsequently, Isherwood, Chen & Mapson (1954) and Mapson, Isherwood & Chen (1954) came to the conclusion from an extensive series of experiments that in plant and animal systems respectively the following sequences were involved: (1) D-Galactose -+ D-galacturonic acid methyl ester -+ L-galactono-y-lactone -+ L-ascorbic acid. (2) D-Glucose -+ D-glucurono-y-lactone -+ Lgulono-y-lactone --. L-ascorbic acid. Hassan & Lehninger (1956) reported that ratliver extracts containing the microsomes and soluble supernatant could convert D-glucuronolactone, L-gulonolactone and also the corresponding free acids into L-ascorbic acid in the presence of cofactors. In experiments previously carried out in this Laboratory it had been observed that rat-liver tissue incubated with sodium pyruvate showed a higher ascorbic acid value than did the controls incubated without the substrate (Roy, Roy & Guha, 1946). But in no experiment was the value so obtained higher than the original ascorbic acid content of the tissue before incubation, and it was possible therefore that pyruvic acid was not a direct precursor. In a search for a system in which a net synthesis over and above the original tissue ascorbic acid level takes place, Chatterjee, Ghosh, Ghosh & Guha (1957a) found, in later experiments, that a marked synthesis of ascorbic acid occurred in homogenates of rat-liver tissue, containing potassium cyanide, from D-glucurono-ylactone, the increase in ascorbic acid value being sometimes two to three times that originally present. No other substrate, including sodium Dglucuronate and sodium pyruvate, could be so converted into ascorbic acid. Subsequently, it was observed that L-gulonolactone, but not sodium L-gulonate, could also act as the substrate and this conversion took place in the absence of cyanide. Liver tissues of all the mammals investigated could bring about this cyanide-stimulated synthesis, but homogenates of the brain (rat), kidney (rat and goat) or adrenal gland (cow) were ineffective in this respect. Extracts from cow and goat livers were found to be the most active, and goat liver, being more freshly available, was chosen for further studies on the enzyme system concemed. The enzyme system was separated from goat-liver homogenates and obtained in the microsomes and the soluble fraction; it could be further concentrated from the extract by precipitation with ammonium sulphate (Chatterjee, Ghosh, Ghosh & Guha, 1958a). The present paper deals with detailed studies of this enzyme preparation.