A new protocol for assaying ornithine decarboxylase activity in whole tissues : use of this protocol to study diurnal ODC rhythms in the liver and thymus of male rats following an acute injection of dexamethasone
Corcoran, Michael J.
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Ornithine decarboxylase (E.C.126.96.36.199, ODC), the initial and rate-limiting enzyme in the synthesis of polyamines, has been used extensively as an indicator in studying: a) RNA, DNA and protein synthesis; b) cell proliferation and differentiation; c) tumorigenesis and carcinogenesis; and d) the induction ability of a variety of hormones in a number of tissues. Measuring enzyme activity using the popular ¹⁴C0₂ trapping method, necessitated development of a protocol eliminating decarboxylation of ornithine via pathways other than ODC. Elimination of "non-ODC" activity was successfully accomplished by employing the following procedures: assaying supernatants from fresh samples immediately; increasing the speed and length of centrifugation; and decreasing the concentration of supernatant in the reaction mixture from 67% to 17%. Comparison of tissue extracts prepared from fresh liver samples to those frozen in liquid nitrogen, showed a 7-fold increase in "apparent ODC" activity in the latter. None of this activity was inhibited by 5mM difluoromethylornithine (DFMO), indicating that sub-sequent decarboxylation could not be attributed to ODC. The developed protocol was employed to study diurnal ODC rhythms in the liver and thymus of male adrenalectomized rats. An acute injection of dexamethasone was administered throughout a 24-hour period to study the influence glucocorticoids have on these rhythms. ODC activity was 3 times higher when dexamethasone was injected at 07:15 a.m., as opposed to 01:15 p.m., 07:15 p.m. or 01:15 a.m. Thymic ODC levels, following injection, were very low regardless of time of administration. Thymic ODC activity was significantly lower (P<0.001) when dexamethasone was administered at 01:15 a.m. in comparison to the other treatment times. Hepatic samples from nonhormone treated animals, showed peak ODC activity when sacrificed at 12:15 a.m. Lowest activity was detected when animals were sacrificed at 07:15 a.m. Thymic tissue from nonhormone treated rats, showed peak activity when animals were sacrificed at 07:15 p.m. and then dropped to markedly low levels by 12:15 a.m.