Regulation of ornithine decarboxylase activity during the Physarum mitotic cycle

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Experimental

REGULATION

Cell Research 107 (1977) 105-l 10

OF ORNITHINE

DURING

THE

PHYSARUM

DECARBOXYLASE MITOTIC

ACTIVITY CYCLE

M. .I. SEDORY and J. L. A. MITCHELL Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA

SUMMARY Omithine decarboxylase (EC 4.1.1.17), the rate-limiting enzyme in polyamine synthesis, appears in two physically and kinetically distinct states in Physarum polycephalum. In this studv. the interconversion between these alternate enzyme forms; which may be-functional in the control of polyamine synthesis, was examined throughout the naturally synchronous mitotic cycle of the multinucleate plasmodia of this myxomycete. By assaying plasmodial samples in selected concentrations of the coenzyme, pyridoxal-5’-phosphate (PLP), it was possible to detail the variations in each of these forms, as well as the pattern of increase in total assayable enzyme, through this cycle. These data suggest that omithine decarboxylase enzyme synthesis is limited to a short period in early to mid S phase, whereas the state of activity of existing enzyme, which is regulated by a post-translational moditication, varies in a more complex pattern. Variations in the active form of this enzyme are found to correlate closely with reported cell cycle changes in cGMP.

The growth of a cell between consecutive cell divisions is not random, but rather a series of precisely ordered chemical changes [l]. It is therefore not surprising that the polyamines such as putrescine, spermidine and spermine, which are frequently associated with macromolecular synthesis, have been found to vary in concentration with respect to defined periods of the mitotic cycle in several cell culture systems [2-4]. Such control over the biosynthesis of the polyamines is thought to be mediated by the regulation of the activity of the initial enzyme, ornithine decarboxylase (ODC, EC 4.1.1.17). Although this enzyme is known to be very sensitive to changes in hormones [5-71, cations [8], cyclic nucleotides [9-111, and other factors which alter growth rate, we do not yet know the regula-

tory mechanism which induces this enzyme to fluctuate sharply at defined periods during a synchronous mitotic cycle such as demonstrated in Don C [12], HTC [3], CHO [ 131, and Chinese hamster V79 cells [2]. The correlation between ODC activity and the mitotic cycle of these cell systems is frequently limited by the artificial methods required to produce and maintain synchronized cultures, the lack of sufficient samples to fully detail rapid activity fluctuations, and our currently insufficient understanding of the regulation of ODC in the mammalian cell. We have been investigating the regulation of ODC activity in the primitive eukaryote, Physarum polycephalum which, because of its precise natural mitotic synchrony and ease of culture, is ideal for such mitotic Esptl

Cd

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cycle studies. Mitchell & Rusch [14] reported unusually great variability in the activity of this enzyme during the progression through this mitotic cycle, variations which suggested a more complex regulatory mechanism than the previously reported periodic synthesis and degradation of this enzyme. Recently this enzyme has been shown to exist in two distinct enzyme forms, which appear to be reversible modifications of the same enzyme protein l-15, 161. One of these (form A) has a high affinity for the coenzyme pyridoxal-5’-phosphate (PLP), increases rapidly in response to growth stimulation, is inactivated very quickly by an inhibitor of protein synthesis, and is closely correlated with changes in the levels of putrescine in vivo [1416]. The second state of this enzyme (form B) requires unphysiologically high concentrations of coenzyme, and appears to be a less active form of this enzyme which is relatively stable in growing cultures [15-161. Since these studies suggest that polyamine synthesis in Physarum is related to the interconversion of this enzyme between these more or less active forms, it is of interest to study the control of this conversion. In the present study we have investigated variations in these ODC forms during the synchronous Physarum mitotic cycle, in an attempt to correlate the control of ODC synthesis and its post-translational activation with events in the mitotic cell cycle. METHODS Surface plasmodia of Physarum polycephalum were prepared by fusing microplasmodia derived from exponentially growing, non-synchronous suspension cultures, as described by Mohberg & Rusch [17]. All cultures were cultivated in the dark at 28°C on the semidefined media of Daniel & Baldwin 1181. The first synchronous nuclear mitosis (MI) occurred J-6 h after the fusion of the microplasmodia. Subsequent mitoses were observed at intervals of 9-11 h. Mitotic phases of the nuclei were observed by phase contrast microscopy, and staged by comparison with the Exprl Ceif Res 107 (1977)

illustrations of Mohberg & Rusch [ 171. The duration of S and G2, and the absence of a Gl period, have been previously reported [ 191. Plasmodial samples were scraped from the filter paper with a spatula, quick frozen in liquid nitrogen, and stored at -20°C until use. Tissue samples were suspended in 3 ml of ice-cold 0.05 M borate buffer (pH 7.8), containing 0.5 mM dithiothreitol and 0.2 mM EDTA, and disrupted by a 30 set sonication. Enzvme activity was determined by adding 0.1 ml of this homogenate to 1.9 ml of 0.05 %l borate buffer (pH 7.8) containing 0.5 mM dithiothreitol, 0.2 mM EDTA, 0.1 mM L-omithine (0.10 j&i) and PLP as indicated. The ‘%02 liberated from this reaction was absorbed in 0.1 ml hyamine hydroxide and counted in a toluene-based scintillation fluid using techniques previously detailed [ 141. The distinction between the enzyme forms, on the basis of a computer analysis of kinetic data, has been completely described elsewhere [16].

RESULTS The proportion of the ODC enzyme in the more active form in Physarum varies markedly with culture age, temperature, media content and rate of growth. To discount the effect such growth conditions may have on the enzyme’s mitotic cycle pattern, two different cultures were used in this study, one having an unusually high proportion of form A enzyme, and another which, due to repeated subculturing, demonstrated slower growth and a depressed proportion of form A ODC. Since the two forms of Physarum ODC are reported [15, 161 to have K, values for PLP of 0.13 and 33 PM, the maximal velocity for samples containing both could be elicited by assaying in the presence of 200 pm PLP, which nearly saturates even the less active form B. As shown in fig. IA, total assayable ODC begins to increase shortly after mitosis and is doubled by the end of the S period. This step pattern is highly reproducible, showing no dependence on the relative proportions of form A and B enzyme. The level of form A ODC enzyme present in these mitotic cycle samples was de-

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Fig. 1. Abscissa: time after mitosis (hours); ordinate: enzyme act. @moles COZ/h~plasmodium). Variations in omithine decarboxylase activity between the second and third synchronous mitosis of a culture demonstrating a low proportion of active enzyme. Samples were assayed as described in Methods, in the presence of either (A) 200 PM or (B) 1 PM PLP. A computer-assisted analysis of the proportion of the total enzyme in the active form was performed on selected samples, as indicated by the bracketed numbers in (B).

termined by assaying in the presence of only 1 PM PLP, a coenzyme concentration which is nearly saturating for the low K,, form A, yet stimulates less than 2% of the form B enzyme. As shown in fig. lB, the plasmodial content of this A form decreases precipitously immediately after mitosis, then increases sharply to a peak in mid S phase and decreases again in late S and early G2. In this particular experiment, the amount of form A ODC did not increase in G2, and therefore did not double before reaching the next mitosis. Such unbalanced growth, noted between the second and third mitosis after macroplasmodial formation,

cycle ornithine

decarboxylase

107

MI1 and MIII, respectively, is consistent with our observation that this set of cultures terminated growth shortly after this third mitosis. The distinction between these ODC forms on the basis of activity at two different levels of coenzyme was supported by a more extensive kinetic analysis of several of these samples, as described previously [16]. In essence, selected samples were assayed over a range of twelve different PLP concentrations producing a non-linear Lineweaver-Burk plot of coenzyme saturation. These curves were analysed by computer to result from the simultaneous reaction of two enzymes acting on the same substrate [20]. The proportion of the total enzyme activity which was due to the low K, A form was calculated, and is indicated, for the respective samples, by numbers in brackets (fig. 1B). Reasonable agreement was obtained between these two methods of distinguishing the A and B forms. As noted in fig. 1B, the net increase in form A enzyme between

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Fig. 2. Abscissa: time after mitosis (hours); ordinate: % of total enzyme activity. Variations in the percent of total activity due to enzyme in the active form. Enzyme samples from various times in the mitotic cycle were assayed in a large range of coenzyme concentrations, and the V, for each enzyme form was calculated by a computerassisted analysis of this kinetic data. The data points each represent the percentage that the computed V, for the low K,,, form is of the total of the V, for both the low and high K, forms.

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prophase and mid-S phase is basically due to the increase in total enzyme present, as the proportion in the A form remains about the same. The decreases in activity t h after metaphase and in G2, however, both result from decreased proportions of total enzyme in the A form, indicating rapid conversion to ODC form B at these times. In the experiment illustrated in fig. 2, each of the mitotic cycle samples was assayed at a variety of coenzyme concentrations, and the relative amounts of the two forms estimated by computer analysis of the curved Lineweaver-Burk plots. Even though essentially 100 % of the ODC of this culture is in the active form before metaphase, almost one half of this is converted to form B enzyme in early S, and reactivated by mid S. During late S, 76 % of this enzyme is again changed to B, with subsequent reactivation occurring during G2. Unlike the experiment illustrated in fig. 1, these cultures did not decrease their rate of growth until after M IV, and they did double the amount of form A enzyme present during the cycle from M II to MIII. Except for the differences noted in G2, both of these experiments, and five other experiments performed on cultures demonstrating intermediate ratios of form A enzyme, illustrate the same pattern of interconversion between the A and B forms of this enzyme. DISCUSSION Total assayable omithine decarboxylase (forms A, B) increases in a step pattern during the Physarum mitotic cycle. Since this enzyme is relatively stable in growth phase Physarum, this pattern suggests that the bulk of this enzyme protein is synthesized within a short period during the S phase. However, not all of this in vitro assayable Exptl

Cell

Res 107 (1977)

ODC has the ability to be active in the low concentrations of PLP found in vivo. In this study we have demonstrated that, although the average amount of this enzyme in the more active A form may vary widely, relative increases and decreases in this form during the mitotic cycle follow a very specific pattern. Form A enzyme is converted to the less active B form during, and immediately following metaphase, and again in mid to late S. Conversion from the B to the A form occurs between 4 and 14 h after metaphase and frequently in early to mid-G2. The changes in activity assayed at 1 PM PLP on borate buffer, which illustrates the available A form resulting from these interconversions, appear to be almost identical to the activity fluctuations reported earlier in Physarum [ 141. Although these earlier studies entailed assays in the presence of 100 JLM PLP, which should have nearly saturated both ODC forms, the Tris buffer used in those experiments has since been found to interfere with ODC activity, especially that requiring elevated coenzyme concentrations. Therefore the pattern produced by assays in the presence of Tris buffer represented the fluctuation in the A form of the enzyme predominantly. Both ODC forms have been shown to produce putrescine in vitro [14, 161, and putrescine levels have been shown to correlate with the activity of the A form during growth stimulation and inhibition. The rapid fluctuations of the A form of ODC during the synchronous mitotic cycle should therefore produce small variations in putrescine levels. Much of the putrescine formed, however, is converted to spermidine and spermine with the aid of the enzyme S-adenosylmethionine decarboxylase, whose activity follows that of the ODC form A. One therefore may expect to find slightly elevated total polyamine-to-protein

Mitotic ratios during mid S and late G2, corresponding to peaks in the ODC A form. Elevated polyamine levels at these times would be consistent with the accumulating evidence that polyamines are involved in DNA synthesis [21] and chromosomal condensation [22]. We were unable to detect small elevations in the polyamine levels using the methods described previously [14], yet Kuehn [23] has observed the expected peak in all three polyamines just before mitosis in Physarum, with a low point in polyamine content about) h after mitosis. The interconversion between the A and B forms of ODC appears to be under extremely tight control as evidenced in fig. 2, where the A form can be seen to change from 100% of the total down to 24%, or up from 50% to lOO%, within a few minutes. As yet, however, the chemical differences between these two forms, as well as the mechanism of interconversion, have not been elucidated. Research on mammalian cell systems has indicated several effector molecules known to vary during the mitotic cycle, which may be important in the general regulation of the activity of ODC. CAMP, for example, stimulates ODC in rat liver [lo], glioma and neuroblastoma cells [ 111, and demonstrates a temporal correlation with the fluctuations of the ODC activity in Chinese hamster V79 cells [2]. In the Physarum cell cycle, CAMP demonstrates only one peak of activity, immediately before mitosis [24]. However, cGMP peaks in mid-S and late G2, with a sharp decrease in level at mitosis and at the end of S, duplicate the pattern of ODC form A exactly. This correlation suggests that cGMP may be necessary for the maintenance of ODC in this more active form. Consistent with this hypothesis is the general observation that periods of rapid cell growth, which normally involve elevated

cycle ornithine

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109

ODC levels, are associated with increased cGMP as opposed to CAMP concentrations. The microtubule system, which shows definite mitotic cycle fluctuations, has also been suggested to be involved in the regulation of ODC activity. Chen et al. [25] has shown that the disruption of microtubules by specific inhibitors appears to be one step in a mechanism which will inhibit ODC activity. The rapid decline in active ODC noted in Physarum immediately after metaphase could, according to this hypothesis, be correlated with the natural dissociation of the microtubules during the dissolution of the mitotic spindle. It is difficult, at this time, to assess the relative importance of the factors discussed above in the regulation of ODC through the mitotic cycle. As yet, we cannot even generalize as to patterns of ODC activity during a typical cell cycle. Activity peaks in late Gl and late S in Don C cells [lo] are temporally similar to the peaks just before and just after S noted in HTC cells [3] and those in Gl and G2+M of CHO cells [13]. Yet in Chinese hamster V79 cells there is only one peak of activity which begins in late Gl and continues through S [2]. The more active A form of Physarum ODC demonstrates peaks similar to those noted in HTC, CHO and Don C cells, but, because Physarum does not have a Gl period, the first peak after mitosis is entirely within early S. As suggested in this study, this pattern of activity results from the combination of the step pattern of discontinuous synthesis of this enzyme protein, and its activation and inactivation, at precise cell cycle times, by reversible, post-translational modification. Precise identification of the biochemical events in the cell’s mitotic cycle which regulate this enzyme depends on future investigations into the mechanism of this modification.

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This research was supported by grants from the NIH (ROI-AMl7949-02) and the Illinois Division of the ACS (76-13).

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