115

Biochimica et Biophysica Acta, 1037 (1990) 115-121 Elsevier BBAPRO 33538

Conformational changes in ornithine decarboxylase enable recognition by antizyme John L.A. Mitchell and Hong J. Chen Department of Biological Sciences, Northern Illinois University, DeKalb, IL (U.S.A.) (Received 3 April 1989) (Revised manuscript received 5 September 1989)

Key words: Antizyme binding; Ornithine decarboxylase structure; Polyamine biosynthesis; Difluoromethylomithine binding

Rapid, polyamine-induced degradation of mammalian ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC) is though to be controlled by the availability of a small, ODC-binding protein termed antizyme. In this study we have investigated the ability of antizyme to bind ODC protein in various altered physiological states. In particular, cold, NaCI, spermidine and deprivation of coenzyme and substrate enhance enzyme-antizyme complex formation and are all found to promote ODC homodimer dissociation. Conversely, conditions that maintain the active ODC homodimer state prevent antizyme binding and inactivation of ODC. Further, covalent modification of ODC near its active site by difluoromethyiornithine or phosphate also increases its sensitivity to antizyme. These results suggest that the initial signal in ODC degradation may actually be a subtle conformational change in the enzyme that enables antizyme to bind to the enzyme and may subsequently facilitate its degradation.

Introduction Ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC) catalyzes the initial step in the biosynthesis of the polyamines, spermidine and spermine, which are essential for cell growth. This enzyme is very sensitively regulated, increasing sharply in activity in response to various growth factors, hormones, carcinogens and mitogens. ODC activity and protein decrease quickly in response to growth inhibition or accumulation of cellular levels of the pathway products, most noticeably spermidine. In mammalian cells the half-life of this enzyme protein can decrease from several hours to 10 min in response to the addition of spermidine to the culture media [1,2]. The mechanism of this polyamine-induced, rapid degradation of ODC is critical to our understanding of the physiological control of polyamine biosynthesis. This also may be an informative model system to help us understand an entire group of control point enzymes that exhibit such extremely short half-lives.

Abbreviations: ODC, ornithine decarboxylase; HTC, rat hepatoma cells; DFMO, difluoromethylornithine. Correspondence: J.L.A. Mitchell, Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, U.S.A.

Antizyme, a small ODC-binding protein, is thought to facilitate the very rapid polyamine-stimulated degradation of ODC [3-5]. Polyamines induce both the synthesis of antizyme and its release from cellular membranes, and its cellular levels change i n a n inverse relationship to active ODC [6]. This protein binds ODC with a very high affinity [7], forming a complex that can be observed during ODC degradation and correlates directly with the half-life of the enzyme [4]. These investigators suggest that the production or release of free antizyme may be the initial rate-limiting step in ODC-antizyme complex formation, and subsequently ODC degradation. If the first step in ODC degradation is the binding of antizyme to the enzyme protein, then in order to comprehend the regulation of this enzyme turnover, it is essential that we understand the elements that control the formation of this complex. Kitani and Fujisawa [7] reported several factors that alter the velocity of ODC interaction with antizyme and speculated that antizyme binding might be influenced by ODC conformation. In the present study we have greatly extended these observations to establish the inability of antizyme to bind to active, dimeric ODC. Further, we demonstrate that, along with factors dissociating the ODC dimer, covalent modification of this protein at or near the catalytically active site can greatly enhance antizyme binding. These rather unexpected observations necessitate a substantial

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

116 revision of our current perception of the role of antizyme in the inactivation of mammalian ODC.

Experimental procedures

Chemicals. Pyridoxal 5'-phosphate, L-ornithine, dithiothreitol, EDTA, morpholinopropanesulfonic acid (Mops), spermidine, and methylbenzethonium hydroxide were purchased from Sigma Chemical Co.. Triton X-100 and Brij-35 were from Pierce Chemical Co. L-[1a4c]Ornithine (61 Ci/mol) was purchased from Amersham/Searle Corp. DL-a-difluoromethyl[3,43H]ornithine (39.2 Ci/mmol) was purchased from New England Nuclear. Monoclonal antibody against antizyme (HZ1B3) was generously provided by Drs. S. Matsufuji and S. Hayashi. Cell culture. Rat hepatoma (HTC) cells were grown in suspension culture in Swim's 77 medium containing 10% (v/v) calf serum (Biologos). The induction of ODC activity and cell harvesting were as previously described [81. Assay of ornithine decarboxylase. The assay for ornithine decarboxylase activity was exactly as described previously [8], except that the buffer used was Mops instead of Epps (n-(2-hydroxyethyl)piperazineN'-3-propanesulfonic acid). Partial purification of ornithine decarboxylase. HTC cell pellets containing (1-3). 108 cells were sonified in 2.0 ml of buffer A (0.02 M Mops (pH 7.2)/0.5 mM EDTA/0.02% Triton X-100/1.0 mM dithiothreitol) containing 1.0/xM pyridoxal 5'-phosphate and 0.25 M NaC1. This was applied to a Sephacryl S-200 column and eluted with the same buffer. The peak activity fractions were pooled, concentrated on a Diaflo YM-30 membrane (Amicon), diluted 5-fold with buffer A, and chromatographed on a Mono Q (Pharmacia) anion-exchange column as described below. Active fractions were pooled and buffer exchanges were made as required using small G-25 columns. In some instances Superose 12 column chromatography either replaced or was used in addition to the Mono Q step. Rapid anion-exchange chromatography. Separation of mammalian cell ODC on the Mono Q, HR 5/5 anionexchange column (Pharmacia)was as described previously [8]. Rapid analytical gel filtration chromatography. Enzyme samples up to 0.2 ml were applied to a Superose 12 (Pharmacia) gel filtration column that had been preequilibrated with buffer as indicated in the figure legends. The enzyme was eluted at 0.4 ml/min into 0.18 or 0.33 ml fractions, as indicated all at room temperature. Standardization of this column was described previously [8]. Preparation of [ 3H]ODC Pellets containing (0.2-1.0) - 108cells were sonified in 1.5 ml of buffer A containing 5/xM pyridoxal 5'-phosphate and 5 mM dithiothreitol.

This was incubated at 37°C for 3.5 h with 50 /~Ci [3H]DFMO, resulting in an 80-95% loss in activity. The sample was then partially purified following the procedure outlined above.

Antibody precipitation of antizyme and antizyme-ODC complexes. Excess (10/xg) monoclonal antibody against antizyme (HZ1B3) was added to 0.2 ml mixtures of ODC and antizyme, and incubated 15 min at 4°C. A mixture (0.04 ml) containing 10 #g rabbit anti-mouse IgG bound with 10% insoluble protein A (Sigma Chemical Co.) was added and incubated at 4°C for 1 h. The precipitate was removed by centrifugation at 10 000 × g for 20 min. Induction and isolation of ODC-antizyme. Suspension cultures of HTC cells at a density of about 1.2.106 were resuspended in fresh media containing 5 mM spermidine. After 4.5 h, the cells were harvested, washed with phosphate-buffered isotonic saline and frozen at - 2 0 °C until use. These pellets were sonified in 2.0 ml of buffer A containing 0.25 M NaC1 and 5.0 mM dithiothreitol, and chromatographed on a Sephacryl S200 column equilibrated with the same buffer. Fractions containing antizyme activity were pooled and concentrated on Diaflo YM-10 membranes (Amicon) and Applied to a Superose 12 gel-filtration column using the same buffer. Active fractions were pooled and diluted 5-fold with buffer A and apphed to the Mono Q anionexchange column and eluted with a 6.0 ml linear gradient of 0.19-0.45 M NaC1 at a flow rate of 1.0 ml/min. 0.15-ml fractions were collected in the middle of the gradient and assayed for antizyme activity. Active fractions were concentrated on a Centricon 10 concentrator (Amicon). Assay of ODC-antizyme. Antizyme was mixed with 0.2-0.5 U of partially purified ODC in 0.2 ml of buffer A containing 0.25 M NaCI (or as indicated). After 10 min incubation on ice, the mixture was assayed for ODC activity. Antizyme activity was determined by measuring the loss of ODC activity attributed to antizyme addition. Inhibition of ODC activity up to 75% was linear with respect to amount of antizyme added. One unit of antizyme activity was defined as the amount inhibiting one unit of ODC.

Results

Correlation between antizyme binding and subunit aggregation Mammalian ODC has been shown to exist predominantly in a homodimer conformation within the intact cell [8,9]. Exposure of isolated enzyme to increasing salt concentrations results in its progressive dissociation to catalytically inactive 55 kDa subunits [9,20]. This dissociation and inactivation can be reversed by supplying coenzyme, PLP [8], and substrate, ornithine [10], to the buffer. Of interest, the conditions that promote enzyme

117 TABLE I Antizyme inactivation of monomer vs. dimer ODC

A preparation of partially purified ODC monomer was split so that half was maintained in the monomer buffer, and the other half was exchanged by G-25 chromatography to ODC dimer-inducing buffer as described in Fig. 1. Multiple aliquots (approx 2.5 U) of the monomer and dimer ODC preparations were than reacted with 2.3 U samples of purified antizyme for 10 rain at 4 ° C. Additions were then made to the mixture in monomer buffer to make it equivalent to the dimer sample, and the dimer sample was diluted with an equivalent amount of dimer buffer. Monoclonal antibody to antizyme (HZ1B3) was then added to the mixtures followed by precipitation with insoluble protein A. In each case samples were removed for ODC assay before and after addition of antizyme to determine the amount of ODC bound and inactivated by the antizyme as a percent of control. The experiment was also repeated without the removal of the ODC-antizyme complex before the assay of remaining ODC activity. Data are presented as units ± standard deviation with n = 4. ODC activity (units)

ODC inactivated

enzyme conformation

without antizyme

with 2.3 units of antizyme

units

fraction of control (~)

Reactions with antibody precipitation of antizyme-complexes ODC

monomer dimer

2.34±0.08 2.36±0.32

0.21±0.08 2.16±0.16

2.12±0.01 0.20±0.17

91.1 7.7

Reactions without precipitation of antizyme-complexed ODC

monomer dimer

2.59±0.05 2.53±0.32

0.35±0.09 2.32±0.16

2.25±0.07 0.22±0.31

86.6 8.0

association and dissociation in vitro are generally recognized as those that respectively stabilize or destabilize the enzyme in vivo. In light of such observations, Kitani and Fujisawa [7] have speculated that salt-induced ODC dissociation may be involved in the reactivity between the antizyme and the enzyme. In order to test this hypothesis it was first necessary to define buffer conditions that would allow extensive control of the state of enzyme polymerization without irreversibly inhibiting enzyme activity. As shown in Fig. 1, enzyme that was partially purified in the monomeric state in buffer containing 0.25 M NaC1 and 1/~M PLP, could be induced to almost 90% dimeric enzyme by placing it in buffer containing less NaC1 (0.1 M), 50/~M PLP and 0.5 mM ornithine. After Superose 12 gel filtration chromatography in the respectively buffers, the two ODC conformations showed equivalent activity in the dimer-promoting assay conditions. Thus, the temporary dissociation of ODC to the monomeric state does not, by itself, inactivate the enzyme. In the experiment shown in Table I, monomer and dimer ODC were prepared from partially purified monomer, as described in Fig. 1, and about 2.4 units of each were reacted with antizyme. Following a 10 min incubation at 4 o C, excess antizyme and ODC-antizyme complexes were removed by precipitation with monoclonal antibody against the antizyme, facilitated by secondary antibody and protein A. Samples of the remaining supernatants were then placed in dimer-inducing assay buffer and the level of active enzyme determined. As expected, the 2.3 units of antizyme added inactivated approx. 2.1 units, or 91% of the monomer form ODC. By contrast, the same amount of antizyme only inactivated 0.196 units, or 7.7% of the dimer enzyme preparation. Since approx. 10% of the

'dimer' enzyme preparation was actually still monomeric, and therefore susceptible to 91% inactivation, it is likely that the 8.3% of the dimer preparation that was inactivated could be attributed to this incomplete dimerization. This suggests that the dimeric ODC state is completely refractory to inactivation by antizyme. In the second set of experiments in Table I the antizyme and antizyme-ODC complexes were not removed prior to sampling the mixtures for remaining ODC activity. This protocol was to test whether or not antizyme that had bound to ODC monomers would dissociate from this complex when the ODC protein was returned to the dimer-inducing conditions of the

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Fr'act ion number" Fig. 1. Analysis of O D C protein conformation using rapid, analytical gel filtration chromatography. Identical samples of partially purified ODC were chromatographed on a 20 ml Superose 12 column in buffer A containing either 0.25 M NaCI and 1.0 #M PLP (o), or 0.1 M. NaC1, 50 #M PLP and 0.5 ornithine (e). Columns were run at 0.4 ml/min and 0.i8 ml fractions were collected. Yeast alcohol dehydrogenase and chicken ovalbumin were used as molecular weight standards. The peaks at fractions 37 and 29 correspond to approximate molecular weights of 55000 and 110000, respectively.

118 assay buffer. As shown in Table I, this exchange does not appear to occur; essentially identical results obtained with or without the ODC-antizyme complex being removed before the enzyme assay. In the above experiments, the equilibrium between association and dissociation of ODC subunits was shifted to favor dimer formation by the addition of both ornithine and PLP to the buffer. As described previously [9], a shift in this equilibrium to favor ODC monomer formation can be achieved by substantial elevation of the NaC1 concentration. Using high-performance gel filtration techniques as in Fig. 1, we have been able to demonstrate that temperature and spermidine levels also influence this equilibrium. By chilling ODC fractions to 4 ° C and chromatographing at this temperature, partial ODC dissociation can be achieved in buffers that are compatible with dimeric ODC at 22 ° C. Similarly, enzyme chromatographed at 22 ° C but with 1.0 mM spermidine added to the buffer underwent complete dissociation to the monomeric state (data not shown).

DFMO inactioation of ODC affects antizyme binding The inactivation of ODC by catalysis with the enzyme-activated irreversible inhibitor difluoromethylornithine (DFMO) is thought to result from covalent addition of the inhibitor to the enzyme in a location that interferes with the active site [11]. Bound D F M O residue does not appear to inhibit antizyme from recognizing ODC, and DFMO-inactivated enzyme has been used to quantitate antizyme-ODC complexes in a simple competition assay in which it replaces the active ODC [5,12-14]. The modification of ODC by D F M O actually may enhance the enzyme's affinity for antizyme. As shown in Fig. 2, when native and DFMO-inactivated enzyme were present in the same reaction tube, added antizyme preferentially bound to the enzyme that had been previously inactivated with DFMO. In this experiment enzyme bound to antizyme was removed by precipitation with excess antibody to antizyme as described above. The enhanced binding of antizyme to DFMO-inactivate ODC may, in part, be associated with enzyme conformational changes induced by the bound D F M O residue. In particular, enzyme inactivated with this inhibitor is measurably defective in its ability to reform the dimeric enzyme conformation from isolated monomers. In the experiment shown in Fig. 3, ODC labeled with [3H]DFMO and an identical preparation of uninhibited enzyme were mixed together in a monomer-inducing buffer, and then changed to dimer-inducing buffer conditions. Subsequent gel filtration chromatography in this dimer buffer revealed that the majority of the native enzyme had regained the dimer conformation at the same time that less than 15% of the DFMO-inhibited enzyme achieved this state. Therefore,

loo o o

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0

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I 90

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Fig. 2. Preferential binding of antizyme to DFMO-inactivated ODC. Partially purified m o n o m e r O D C (3.1 U) and O D C (3.4 U) inactivated with [3H] D F M O were mixed in buffer A containing 2.0 /tM PLP and without NaCl or ornithine. These conditions allow partial O D C dimer formation. Purified antizyme (0.6 U / 3 0 #l) was added as indicated and the mixture was reacted at 4 ° C for 10 rain before precipitation with excess (40 #g) monoclonal antibody (HZIB3) to antizyme and 10% protein A. Unprecipitated O D C activity ( I ) and [3H]ODC (n) were analyzed and plotted as percent of control.

the preferential binding of DFMO-inactivated ODC by antizyme, in experiments conducted as in Fig. 2, may be related to the diminished ability of this modified enzyme to go from the antizyme sensitive monomeric state to the antizyme insensitive dimer condition.

O. 4

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35

40

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number

Fig. 3. Decreased propensity of DFMO-inactivated O D C to assemble

to the homodimer conformation. Partially purified fractions of active and [3H]DFMOinactivated ODC monomers were prepared as described in Experimentalprocedures. These were mixed and the buffer exchanged, using G-25 chromatography, to a dimer-inducing buffer (buffer A with 0.1 mM NaC1, 50/xM PLP, 0.1 mM ornithine, and 0.5 mM each of CaCI2, MgCI2 and MnCI2). This enzyme mixture was then chromatographed on a Superose 12 column at 0.4 ml/min at room temperature. The fractions collected(0.33 ml) were assayed for both ODC activity (@) and 3H counts (o). Fractions 16 and 20 correspond to approximate molecular weights of 110000 and 55000, respectively.

119

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Fig. 4. Preferential binding of antizyme to ODC-II. Pellets of HTC cells containing approximately equal amounts of ODC-I and II were homogenized in buffer A containing 10 mM K2PO4 and 1.0 mM sodium vanadate (to inhibit ODC II to I conversion). Equal portions were incubated for 10 min at 4 ° C with (o) or without (Q) sufficient purified antizyme to inactivate approximately 50% of the enzyme. Excess monoclonal antibody HZ1B3 was then added to these mixtures in order to precipitate enzyme-antizyme complex and any free antizyme, and the antibody complexes were removed with the aid of 10% insoluble protein A. The supernatants were subsequently chromatographed on a Mono Q column as described in Experimental procedures. The eluted fractions were assayed for ODC activity and the amounts of ODC-1 (fractions 7-15) and ODC-II (fractions 17-23) remaining after antizyme treatment were compared with the untreated controls.

Correlation between ODC charge state and antizyme binding ODC isolated from mammalian cells, like that of several other eukaryotes, can be separated by ion-exchange chromatography or isoelectric focusing into two distinct charge forms. Conversion of the more negatively charged form, ODC-II, to ODC-I by various phosphatases suggests that these isoforms of the enzyme result from a post-translational phosphorylation [13,15]. Although this modification does not alter the catalytic properties of the enzyme [16], it does appear to have an effect on its stability within the intact cell [2,12,17]. Since ODC degradation is thought to be related to its interaction with antizyme, it is of interest to know whether a correlation exists between the charge state of the enzyme and its ability to be inactivated by antizyme. In our initial investigation of this question we demonstrated that antizyme can inactivate both ODC-I and II, yet we were unable to detect any difference in their relative affinity for this regulatory protein [12]. As shown in Fig. 4, however, a slight preference for the more negatively charged state, ODC-II is apparent when the interaction between enzyme and antizyme is limited to a short exposure. In this study a crude homogenate containing ODC of both charge states was maintained

at 4 ° C in a buffer that induced partial dissociation of the enzyme dimers. Antizyme sufficient to inactivate 50% of the enzyme was incubated with this homogenate for 10 min. Then antizyme-bound enzyme, along with any excess antizyme, was removed by precipitation with antibody specific for the antizyme. The ODC that was not precipitated along with the antizyme was then immediately chromatographed on the Mono Q column to determine loss of each enzyme form relative to unreacted controls. In the typical result shown, there was a 70.9% loss in ODC-II compared to a 39.3% decrease in ODC-I. In the course of six similar repeats of this experiment we observed an average of 1.79-times as much loss of ODC-II as I. Discussion This study revealed that even minor alterations in ODC structure have a major influence on the binding of antizyme. The marked specificity of antizyme for the monomer, in preference to the dimer, form of ODC greatly extends the observations made by Kitani and Fujisawa [7]. These authors demonstrated that the velocity of the antizyme binding to ODC was increased by elevating the NaC1 concentration of the buffer and decreasing the temperature of the reaction. From the present studies it appears that the enhanced antizyme binding results from the fact that these changes in temperature and salt concentration promote dissociation of the ODC dimer to the antizyme-sensitive monomer conformation. The fact that Kitani and Fujisawa [7] still observed some inhibition of ODC when antizyme was added in the absence of NaC1 at 3 7 ° C suggests that partial dissociation of ODC was still occurring. In this regard it should be noted that they did not use omithine in their buffer, and they did use a phosphate buffer which has previously been demonstrated to favor ODC dissociation even in the presence of coenzyme and substrate [9,18]. The fact that antizyme does not bind to dimeric ODC is of great significance because of the implication this has on enzyme-antizyme complex formation, and thereby ODC turnover, within the intact cell. In induced tissues, ODC is found to be in its active, dimer conformation [8], a form our data suggest is refractive to antizyme binding. Consistent with this model there are several instances where antizyme has been found in both free and complexed states where stable, active ODC is also present [3,5,19-22]. However, if cytoplasmic levels of ornithine or PLP were to decrease or levels of spermidine to increase, in the cytoplasm surrounding the O D C molecules then, as has been shown here and by others [8-10], ODC dissociation could be promoted. This, in turn, could allow antizyme to bind and perhaps signal the start of ODC protein degradation. In support of this model, ODC instability has been

120 associated with physiological changes that facilitate enzyme dissociation such as a limitation of coenzyme availability or an increase in the media osmotic strength. Of particular interest, the pathway product spermidine is known to greatly shorten the half-life of O D C through an unknown indirect mechanism [1,2]. This polyamine has now been shown to induce monomer formation of mammalian ODC. It therefore is possible that spermidine affects O D C turnover not only by stimulating the synthesis of antizyme but also by promoting enzyme dissociation to an antizyme-sensitive conformation. Inactivation of O D C by catalysis with D F M O results in the covalent linkage of the D F M O residue somewhere near the enzyme active site [23]. Although initial reports suggested that this modification interfered with the enzyme's ability to associate with antizyme [24], subsequent studies have shown that DFMO-inactivated enzyme does not prevent antizyme binding to O D C [5,12,21]. Furthermore, the latter workers have utilized the ability of DFMO-inactivated O D C to displace native ODC from ODC-Az complexes in order to quantify these complexes in homogenates of tissues [5,12,21,22]. In this report we have presented data that suggest that the proficiency of this replacement assay may be partially attributed to the fact that antizyme preferentially binds the DFMO-inactivated enzyme. Although this is beneficial in the displacement assay, such preferential treatment of DFMO-inactivated enzyme by antizyme could seriously alter the interpretation of m a n y in vivo and in vitro experiments that use labeled D F M O to ' tag' ODC. Up to now the implicit assumption has been that ODC labeled with the [3H]- or [14C]DFMO is equivalent to the native enzyme. The third O D C modification tested for its influence on antizyme binding was the phosphorylation that distinguishes O D C - I I from O D C - I [8,13,15]. As previously demonstrated [12], both charge states of the enzyme can bind antizyme; however, in this study a preference was observed for ODC-II. The preferential binding of the phosphorylated enzyme form is slight, and probably only detected in these experiments because of major improvements in the experimental technique, including the removai of the antizyme-bound O D C before chromatography and the use of very rapid, high-performance ion-exchange chromatography. The selectivity of antizyme for O D C - I I is somewhat puzzling in that antizyme binding is though to be associated with enzyme inactivation and O D C - I is generally considered to be the most labile form within the intact cell [12,25]. The reason for this preferential binding is not yet clear, but since we have not detected any difference in ability of these enzyme charge states to reform active enzyme dimers (unpublished data), other effects of this charge difference on the enzyme conformation must be considered. In these studies several modifications of the O D C

protein were found to alter this enzyme's affinity for the regulatory protein, antizyme. F r o m these observations, inferences can be made as to the structural regions of O D C that are recognized by this peptide. In particular the binding site appears to be hidden in the intact O D C dimer, suggesting that the antizyme recognizes the m o n o m e r surface involved in O D C dimerization. Further, recognition may be in the proximity of the O D C catalytic site, as bound remnants of the inhibitor D F M O may also alter the binding of antizyme. Finally, the recognition site may also overlap a residue presumed to be phosphorylated in ODC-II. These observations alter our concept of the role antizyme plays in O D C inactivation. In the past it was assumed that active O D C could not coexist with active, free antizyme in the intact cell because of the extremely favorable binding constant between these proteins [7]. Now the possibility must be considered that these proteins do coexist and that the pathway products (e.g., spermidine), coenzyme, substrate or covalent modifications may control antizyme-ODC complex formation, and thereby O D C degradation, merely by shifting the enzyme m o n o m e r - d i m e r equilibrium.

Acknowledgments This work was supported by Research Grant G M 33841 and B R S G R R 07176 from the National Institutes of Health.

References 1 Holtta, E. and Pohjanpelto, P. (1986) J. Biol. Chem. 261, 9502-9508. 2 Mitchell, J.L.A., Mahan, D.W., McCann, P.P. and Qasba, P. (1985) Biochim. Biophys. Acta 840, 309-316. 3 Hayashi, S.I., Kameji, T., Fujita, K., Murakami, Y., Kanamoto, R., Utsunomiya, K., Matsufuji, S., Takiguchi, M., Mori, M. and Tatibana, M. (1985) Adv. Enzyme Reg. 23, 311-329. 4 Murakami, Y. and Hayashi, S. (1985) Biochem. J. 226, 893-896. 5 Murakami, Y., Fujita, K., Kameji, T. and Hayashi, S. (1985) Biochem. J. 225, 689-697. 6 Heller, J.S. and Canellakis, E.S. (1981) J. Cell. Physiol. 107, 209-217. 7 Kitani, T. and Fujisawa, H. (1985) Biochem. Int. 10, 435-440. 8 Mitchell, J.L.A., Rynning, M.D., Chen, H.J. and Hicks, M.F. (1988) Arch. Biochem. Biophys. 260, 585-594. 9 Kitani, T. and Fujisawa, H. (1984) Biochim. Biophys. Acta 784, 164-167. 10 Solano, F., Penafiel, R., Solano, M.E. and Lazano, J.A. (1985) FEBS Lett. 190, 324-328. 11 Pegg, A.E., McGovern, K.A. and Wiest, L. (1987) Biochem.J. 241, 305-307. 12 Mitchell, J.L.A., Qasba, P. and Mahan, D.W. (1985) in Recent Progress in Polyamine Research (Selmeci, L., Brosnan, M.E. and Seiler, N., eds.), pp. 55-64, Akademiai Kiado, Budapest. 13 Mitchell, J.L.A., Hicks, M.F., Hoff, J.A. and Chen, H.J. (1989) Adv. Exp. Med. Biol. 250, 55-70. 14 Flamigni, F., Stefanelli, C., Guarnieri, C. and Caldarera, C.M. (1986) Biochim. Biophys. Acta 882, 377-383.

121 15 Peng, T. and Richards, J.F. (1988) Biochem. Biophys. Res. Commun. 153, 135-141. 16 Mitchell, J.L.A. and Mitchell, G.K. (1982) Biochem. Biophys. Res. Commun. 105, 1189-1197. 17 Pereira, M.A., Savage, R.E. and Guion, C. (1983) Biochem. Pharmacol. 32, 2511-2514. 18 Mitchell, J.L.A., Carter, D.D. and Rybski, J.A. (1978) Eur. J. Biochem. 92, 325-331. 19 Laitinen, P.H. (1985) J. Neurochem. 45, 1303-1307. 20 Laitinen, P.H., Hietala, O.A., Pulkka, A.E. and Pajunen, A.E.I. (1986) Biochem. J. 236, 613-616.

21 Flamigni, F., Stefanelli, C., Guarnieri, C. and Caldarera, C.M. (1986) Biochim. Biophys. Acta 882, 377-383. 22 Onoue, H., Matsufuji, S., Nishiyama, M., Murakami, Y. and Hayashi, S.I. (1988) Biochem. J. 250, 797-803. 23 Metcalf, B.W., Bey, P., Danzin, C., Jung, M.J., Casara, P. and Vevert, J.P. (1978) J. Am. Chem. Soc. 100, 2551-2553. 24 Kyriakidis, D.A., Flamigni, F., Pawlak, J.W. and Canellakis, E.S. (1984) Biochem. Pharmacol. 33, 1575-1578. 25 Mitchell, J.L.A., Qasba, P., Stoiko, R.E. and Franzen, M.A. (1985) Biochem. J. 228, 297-308.