Indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine in rat hepatoma cells

Abstract

Direct or indirect inhibitors of L-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to L-ornithine, including DL-.alpha.-difluoromethylornithine, .alpha.-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-L-methionine (SAM) decarboxylase activity (EC 4.1.1.50). Comparison of the catalytic properties of SAM from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis(guanylhydrazone). Actinomycin D and cycloheximide an blocked the increase of SAM decarboxylase activity elicited by .alpha.-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated SAM decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. Elevation of SAM decarboxylase activity by .alpha.-difluoromethylornithine may be due to stabilization of the enzyme. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased SAM decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and SAM S decarboxylase activity could be established. Addition of 1 mM-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of SAM decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with .alpha.-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10 .mu.M) was as effective as spermidine in decreasing SAM S decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. The spermidine site of action appears to be post-translational since the spermidine-induced decrease of SAM activity was not prevented by actinomycin D and spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of SAM decarboxylase by spermidine. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of SAM decarboxylase is discussed.