The brains of adult male rats were dissected to five regions: cerebellum, cortex, hippocampus, hypothalamus and "midbrain". When crude cytosol fractions or ammonium sulphate-precipitated dialysates were assayed for ODC activity, the hypothalamus and midbrain showed highest activity and the cerebellum lowest. However, the difference was less than 10-fold. The presence of 0.1 mM GTP in the enzyme assay increased ODC activity in the cerebellum lysate nearly two-fold, increases in other lysates were minor and activity even decreased in the dialysed (NH4)2SO4-precipitate of hippocampus lysate (0.82-fold change).
In order to further characterize the ODC activities of brain regions, gel-permeation chromatography in the presence of 0.25 M NaCl was performed to dissociate the ODCantizyme complex and to separate the ODC and the antizyme from each other. The GTPactivatable ODC activity was clearly found from each brain regions in the eluted fractions. In the cerebellum ODC activity was increased 4.2-fold in the presence of 0.1 M GTP, in all other regions increases were between 2.0- and 2.7 fold. The specificity of activation was tested by incubating the brain and kidney enzymes with various nucleotide triphosphates. As expected, kidney ODC was not affected by any of nucleotides tested. However, the ODCs of midbrain and hypothalamus that were chosen to represent brain ODC, were stimulated 1.5 – 2-fold by GTP, dGTP and ATP, and slightly by dATP. The pyrimidine nucleotides were ineffective. The K1/2 values for GTP and ATP were approximately 2 µM and 40 µM, respectively, indicating that GTP was favoured over ATP and could activate brain ODC in lower concentration. The GTP-induced activation of ODC was essentially irreversible since activity was elevated even after GTP was removed from the preincubated lysates by extensive dialysis. GTP appeared to effect on ODC mainly by increasing Vmax value of the enzyme.
Because the GTP-activation of ODC could be detected clearly only when the enzyme was dissociated and separated from the antizyme, we studied the sensitivity of brain and kidney ODCs for the inhibition by antizyme. A constant amount of ODC activity was mixed with increasing amount of partially purified brain antizyme and the ODC activity remaining was assayed. There was no difference in the inhibition of the kidney ODC in the presence or absence of GTP, whereas the GTP-activatable brain ODC activity from the gel filtration fractions of the cerebellum appeared to be more sensitive to antizyme inhibition than activity assayed in the absence of GTP. When sensitivity to heat inactivation was studied, brain ODC activity was more stable than activity in kidneys. However, GTP did not have any significant effect on the heat sensitivity of brain ODC.
Since it has been suggested that the basal ODC activity in the neocortex and the cerebellum is resistant to DFMO (Zawia et al. 1991), we determined the effect of DFMO on the ODC activity in the different brain regions. DFMO inhibited around 60 % of the ODC activity from all brain regions. However, the activities detected after extensive dialysis were very low both in the controls and in the DFMO inhibited samples. Kidney ODC was inhibited virtually completely.