Physiological and biochemical differences between the two charged forms of mammalian ornithine decarboxylase
Ornithine decarboxylase (ODC; E.C. 22.214.171.124), is the rate-limiting enzyme of the polyamine biosynthetic pathway in eukaryotic cells. The enzyme is distinguished by having the shortest half-life of any known mammalian enzyme. ODC of rat hepatoma (HTC) cells exist basically in two charged states, ODC-I and ODC-II, that are separable on a DEAE ion-exchange column. This charge difference between the enzyme forms is maintained in partially purified preparation. Very little is known about the function of these altered states of the enzyme and how distinct these forms are under varied physiological and biochemical conditions. In this project an attempt has been made to elucidate any physiological or biochemical distinctions between the ODC forms. The ODC forms were prepared from homogenates of HTC cells and examined for various physical characteristics and distinct physiological activities. Physically the two major forms were found to be quite similar. Molecular weight, thermal sensitivity, location within the cell and the optimal hydrogen ion concentration studies demonstrated similarities between these forms. Minor differences were noted in that form I appears to be slightly more sensitive than the others to heat, and form III shows a different pH optima than I and II. These forms were also shown to be closely related by the observation that form II readily shifts to form I in crude homogenates. This observation was confirmed by studies using ³H-difluromethylornithine (DFMO), an enzyme- activated irreversible inhibitor of ODC, to follow the physical change in this enzyme's charge. The physiological activities of these forms are quite similar also. They appear to interact with the ODC-antizyme, in vivo and in vitro with equal affinity. Interaction of these forms with DFMO also shows no specificity. Yet, within the cell they appear to behave separately. Cycloheximide studies demonstrate that the half-lives of the two forms are distinct in that form I appears to be more labile than II. It therefore appears that the two forms act as separate pools of enzyme and these pools appear to be physically modified into one or the other form. These forms are similar proteins, essentially one enzyme and a single gene product with a possible post-translational modification resulting in the two forms.