Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

Abstract

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.

High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.

“…In data not shown, the specific activity for the recombinant ODC was determined by assaying the level of binding of radioactive DFMO (a suicide inhibitor of ODC) to protein in the soluble fraction of bacteria transformed with pBAcdc (36). Depending on whether one assumes that one molecule of DFMO is covalently bound to each subunit (37) or to each dimer of the enzyme, the binding assay indicated that the specific activity for the recombinant enzyme was two-thirds to one-third, respectively, that for the native enzyme purified from trypanosomes [at 50,000 nmol min-l.mg-1 (12) While essentially all of the HPRT produced by pBSprt is in the soluble fraction (Fig. 3 lane 6), SDS gel analysis of the proteins from bacteria containing the pBAcdc plasmid showed that the yield of soluble ODC was optimal if the bacteria were cultured at 30°C rather than at 37°C.…”

Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.

“…All the variants had increased ODC activity, and in all the variants the activity was modulated by experimental manipulation of polyamine pools. We did not measure the number of ODC molecules in extracts, but it is a reasonable presumption, based on the work of others (28,29), that activity reflects the amount of enzyme protein. All variants share an increase in the amount of ODC mRNA compared with that present in wild-type cells.…”

Novel properties of malarial S-adenosylmethionine decarboxylase as revealed by structural modelling

“…Success to date has been moderate, generally it is only possible to induce cytostasis [8]. This is thought to be partly due to the short half-lives of the main regulatory enzymes [3,9]. However, one polyamine targeting drug, 2-difluoromethylornithine (DFMO), has already been used to successfully treat African sleeping sickness caused by Trypanosoma brucei gambiense [10,11].…”