Cysteine liver slices

M. . Krahl Dr. Anfinsen mentioned an attempt to test the possibility that GSH may serve as a source of amino acid for protein synthesis. There may be a n unber of other people who have done negative experiments of this sort. I am one of them. With S Mabeled cysteine and S -labeled GSH, which were kindly supplied by Dr. Tabern and Dr. Hostattler, I measured the amount of S activity incorporated into a crude protein fraction by liver slices in vitro. The molar concentration of cysteine and GSH in the incubation medium was the same in the two respective experiments and the protein fraction was one which had been put through performic-acid treatment. We presume that whatever S was there was not merely attached to the protein via —S—S— linkages. The S was incorporated into this protein fraction 10 times as rapidly from cysteine as it was from GSH. This may mean quite a few things, but gives no support, unfortunately, to the idea that GSH may be an intermediate between cysteine as an amino acid and cysteine in peptide linkage in protein. 

Corroborative evidence has been obtained from experiments with isolated tissue preparations. The in vitro formation of cystine by liver slices (and a saline extract) was observed in nuxtures containing dl-homocysteine and DL-serine. Neither substance was effective when incubated alone. The L-forms of the two amino acids were implicated in the reaction, as D-homocysteine and o-serine did not substitute for the DL-forms and the isolated cystine had the L-configuration. The reaction proceeded best under anaerobic conditions and in the present of O.OOlAf CN , as the latter inhibits cysteine desulfhydrase activity. Methionine substituted very poorly for homocysteine in vitro. 

Whatever the mechanism of cysteine reduction, it is evident that, if the above reaction takes place, cystine metabolism approaches cysteine metabolism. This is admitted by Pirie (96) in his scheme to illustrate the oxidation of cystine to sulfate by rat liver slices ... 

Tissue extracts which exhibit little acetylation in contrast to slices actively deacetylate mercapturic acids (Bray and James, 19f30). Kidney extracts are more active than liver. The deacetylase content of extracts of rat, rabbit, and guinea pig liver were roughly equivalent but varied with the type of substituent on the sulfur. For example, the rabbit dc-acetylase activity toward jV-acetyl-S-benzyl-L-cysteine was low, w hereas toward iV-acetyI- S-butylcysteine it w as high. Bray and James concluded that there is little to support the view that tiie failure of the guinea pig to excrete significant amounts of mercapturic acids is due to the deacetylase activity of its tissues. Probably the failure is in part in the acetylation process and in part due to convemion of the S-substituted cysteines into other metabolism products. 

Pirie (95) used slices of liver and kidneys of rats and found that these tissues oxidize the cysteine sulfur to sulfate. Having thus shown the enzymic character of this oxidation, he attributed it to the existence of a cysteine oxidase. This substance is lacking in blood, testicles, spleen, heart, and lung. The same enzymic reaction was observed by Medes (85) with a filtrate of ground rat liver in a solution buffered with carbonate. The enzymic system under observation, suspended either in a carbonate buffer solution, or in a dilute neutral salt solution, is easily adsorbed on permutite, but its subsequent elution has not been accomplished to date. Otherwise, the same enzymic system is precipitated upon addition of magnesium or sodium sulfate, but it then loses all its activity. 

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