Purification of ornithine decarboxylase from kidneys of androgen-treated mice.
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.
D000728:Androgens / Q000494:pharmacology*; D000818:Animals; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D004229:Dithiothreitol / Q000494:pharmacology; D000518:Eflornithine; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D009952:Ornithine / Q000031:analogs & derivatives / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000302:isolation & purification*; D011732:Pyridoxal Phosphate / Q000378:metabolism
D000728:Androgens; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D009952:Ornithine; D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase; D004229:Dithiothreitol; D000518:Eflornithine