ODC activity was assayed by measuring the release of CO2 from [1-14C]-ornithine (Amersham International, Buckinghamshire, UK) essentially as described (Jänne & Williams-Ashman 1971). The reaction mixture contained 12.5 mM Tris-HCl buffer, pH 7.3, 1 mM EDTA, 2.5 mM DTT, 0.2 mM PLP and 15 µM L-ornithine (1.25 nmol of [1- 14C]-ornithine). Kinetic parameters were determined from Lineweaver-Burk reciprocal plots (I, II). In the kinetic analysis the concentration of L-ornithine varied from 0.05 mM to 1 mM. To determine the sensitivity to heat inactivation, tissue lysates were preincubated at 55 ¢ªC (I). To measure the effect of DFMO inhibition the samples were incubated with either L-ornithine (1.25 mM) or DFMO (2.5 mM) for one hour at 37 ¢ªC, then dialysed overnight against a 2000-fold excess of the reaction mixture buffer without L-ornithine and assayed for ODC activity (I, II).
Antizyme activities (I) were determined as described (Fong et al. 1976). The principle of the assay was to mix the antizyme sample with an ODC preparation of known activity, and to compare the observed enzyme activity in the mixture with the expected value.