No abstract

Ornithine decarboxylase has been purified to homogeneity from kidneys of androgen-treated mice. Such kidneys have an enzyme content 2 orders of magnitude greater than that of other mammalian tissues such as induced rat liver, and only a 10350-fold purification was needed for purification. The enzyme preparation gave a single band on isoelectric focusing and on polyacrylamide gel electrophoresis under native and denaturing conditions. These bands corresponded to the enzyme activity and to the migration of ornithine decarboxylase labeled by reaction with alpha-(difluoromethyl) [5-14C]ornithine, a specific inhibitor. The enzyme has a Mr of about 100 000 and is a dimer of subunit Mr 53 000. The Km for L-ornithine was 75 micron and for pyridoxal phosphate, 0.3 micron. The preparation had a specific activity of 50 mumol of CO2 produced min-1 mg-1 and bound a stoichiometric amount of the irreversible inhibitor, alpha-(difluoromethyl)ornithine (one molecule per subunit). The purified enzyme was unstable even in the presence of 2.5 mM dithiothreitol and 40 micron pyridoxal phosphate unless 0.02% Brij 35 was added. In the presence of this detergent, the enzyme could be stored with little loss of activity.

No abstract

1. The content of decarboxylated S-adenosylmethionine (AdoMet) in transformed mouse fibroblasts (SV-3T3 cells) was increased 500-fold to about 0.4fmol/cell when ornithine decarboxylase was inhibited by α-difluoromethylornithine. This increase was due to the absence of putrescine and spermidine, which serve as substrates for aminopropyltransferases with decarboxylated AdoMet as an aminopropyl donor, and to the enhanced activity of AdoMet decarboxylase brought about by depletion of spermidine. The increase in decarboxylated AdoMet content was abolished by addition of putrescine, but not by 1,3-diaminopropane. 2. 5′-Methylthiotubercidin also increased decarboxylated AdoMet content, presumably by direct inhibition of aminopropyl-transferase activities, but the increase in its content and the decline in spermidine content were much less than those produced by α-difluoromethylornithine. 3. Decarboxylated AdoMet content of regenerating rat liver was measured in rats treated with inhibitors of ornithine decarboxylase. The content was increased by 60% 32h after partial hepatectomy in control rats, by 90% when α-difluoromethylornithine was given to the partially hepatectomized rats, and by 330% when 1,3-diaminopropane was used to inhibit putrescine and spermidine synthesis. After 48h of exposure to 1,3-diaminopropane, which completely prevented the increase in spermidine after partial hepatectomy, there was a 5-fold rise in hepatic decarboxylated AdoMet concentration. These increases were prevented by treatment with putrescine or with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase. 4. These results show that changes in AdoMet metabolism result from the administration of specific inhibitors of polyamine synthesis. The possible consequences of the accumulation of decarboxylated AdoMet, which could, for example, interfere with normal cellular methylation or lead to depletion of cellular adenine nucleotides, should be considered in the interpretation of results obtained with such inhibitors.