Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled alpha-difluoromethylornithine.
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.
D000818:Animals; D002262:Carboxy-Lyases / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003959:Diamines / Q000494:pharmacology; D000518:Eflornithine; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects / Q000201:enzymology; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D014018:Tissue Distribution
D003959:Diamines; D065108:Ornithine Decarboxylase Inhibitors; D003513:Cycloheximide; C009475:trimethylenediamine; D009952:Ornithine; D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase; D000518:Eflornithine