Regulation of S-Adenosylmethionine Decarboxylase Activity in Rat Liver and Prostate*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 260, No. 17, Issue of August 15, pp , 1985 Printed in U.S.A. Regulation of S-Adenosylmethionine Decarboxylase Activity in Rat and * (Received for publication, January 11, 1985) Akira Shirahata and Anthony E. PeggS From the DeDartment of Physiology. Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania Two methods were used for the quantitation of S- enzyme in the biosynthesis of the polyamines, spermidine and adenosylmethionine decarboxylase protein. The first spermine (1-4). In mammalian cells the concentration of involved titrating the active site of the enzyme by decarboxylated AdoMet is normally very low and is a limiting reduction of the Schiff base between H-decarboxyl- factor in the production of polyamines (4, 5). Mammalian ated S-adenosylmethionine and the pyruvate pros- AdoMet decarboxylase is activated by putrescine (2, 3), and thetic group with sodium cyanoborohydride. The sec- this activation probably plays an important physiological role ond method was radioimmunoassay with rabbit anti- in coordinating the availability of putrescine and decarboxserum which was used to determine the total immuno- ylated AdoMet, the two substrates of spermidine synthase. reactive enzyme protein. It was found that the in- However, even when in vitro assays are conducted in the creased S-adenosylmethionine decarboxylase activity presence of saturating concentrations of putrescine, extracts produced in rat prostate by treatment with a-difluo- from cells treated with various pharmacological or physiologromethylornithine and in both prostate and liver by ical agents show striking changes in AdoMet decarboxylase methylglyoxal bis(guany1hydrazone) were due entirely activity (reviewed in Ref. 3). Of particular interest are the to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either increases in AdoMet decarboxylase activity in response to method) remained constant in rats treated with the inhibitors of polyamine synthesisuch as methylglyoxal drugs. Treatment with 2% a-difluoromethylornithine bis(guany1hydrazone) (), a-difluoromethylornithine in the drinking water 3 for days increased prostatic S- (DFMO), and S-adenosyl-1,8-diamino-3-thiooctane and the adenosylmethionine decarboxylase protein by &fold. decrease in response to exogenous polyamines (2-9). These A substantial part, but not all, of this increase could be studies indicate that AdoMet decarboxylase activity is very accounted for by a slowing of the rate of degradation closely regulated by the polyamines, and this regulation acts of the enzyme. The half-life for loss of activity and to ameliorate the effects of polyamine biosynthesis inhibition. titratable protein after inhibition of protein synthesis Such inhibitors have considerable therapeutic potential (10, by cycloheximide was increased from 35 to 108 min ll), and an understanding of the mechanism by which comby treatment with a-difluoromethylornithine. How- pensatory increases in AdoMet decarboxylase are brought ever, the half-life for loss of immunoreactive protein about may assist in the design of appropriate protocols. which was considerably longer was only increased Even in tissues such as rat prostate which have relatively from 139 to 213 min. The molecular weight of the S- high AdoMet decarboxylase activities, the amount of the adenosylmethionine decarboxylase subunit determined enzyme protein is small (12), and it has been difficult to by immunoblotting was 32,000, and no smaller im- develop procedures to quantitate it. Antibodies have been munoreactive fragments were detected. These results raised to purified AdoMet decarboxylase preparations, but indicate that spermidine depletion produced by a-di- only semiquantitative determinations based on immunotitrafluoromethylornithine affects the degradation of S- tion of the activity have been reported (13-16). In the present adenosylmethionine decarboxylase at an early step inwork, AdoMet decarboxylase protein has been determined by volving the loss of the active site without substantial breakdown of the protein. two separate methods. First, the active enzyme has been titrated by the addition of labeled decarboxylated AdoMet and sodium cyanoborohydride (17-19). This procedure inactivates the enzyme by reducing the azomethine bond between S-Adenosylmethionine (AdoMet ) decarboxylase is a key the reaction product and the enzyme s pyruvate prosthetic group and thus leads to a stoichiometric addition of the nucleoside. Second, the total amount of AdoMet decarboxylase protein has been assayed using a competitive radioim- * This research was supported by Research Grant CA18138 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Oduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The abbreviations used are: AdoMet, S-adenosylmethionine; AdoMetDC, S-adenosylmethionine decarboxylase;, methylglyoxal bis(guany1hydrazone); DFMO, u-difluoromethylornithine; RIA, radioimmunoassay munoassay. The results indicate that changes in AdoMet decarboxylase activity in response to DFMO, exogenous polyamines, and are due to parallel changes in the amount of enzyme protein, and that part of the increase in AdoMet decarboxylase activity caused by DFMO is due to a reduced rate of degradation of the protein. Evidence that degradation of the enzyme proceeds via an initial step inactivating the active center prior to loss of immunoreactive protein was also obtained.

2 9584 Regulation of S-Adenosylmethionine Decarboxylase DAYS FIG. 1. Effect of DFMO on prostatic AdoMet decarboxylase. Control Rats were treated with DFMO (2% in drinking water) for the time shown and prostate extracts prepared and analyzed for AdoMet DFMO (2%) decarboxylase activity (0). The content of AdoMet decarboxylase protein was also determined by either RIA (W) or titration with DFMO (2%) + decarboxylated AdoMet (A), and the ratio of activity to protein is i.p.b injecplotted. Results are shown f S.E. for 4 rats at each point. tion (80 EXPERIMENTAL PROCEDURES AND RESULTS~ mg/kg) DFMO (2%) + Rats treated with DFMO by replacement of the drinking water with a 2% solution of DFMO showed a substantial rise (80 mg/kg) in the activity of prostatic AdoMet decarboxylase (Fig. 1). ND, not determined. 6. Activity was increased more than %fold within 1 day, and a ~p., intraperitoneal. maximal &fold increase occurred after 3 days of exposure. The increased activity of AdoMet decarboxylase was paralleled by an increase in the amount of immunoreactive protein (measured by RIA) and by an increase in the amount of active AdoMet decarboxylase measured by titration of the active site with decarboxylated AdoMet in the presence of sodium cyanoborohydride (Fig. 1). Full details of these procedures are Treatment Tissue given in the Miniprint Supplement. Within the limits of experimental error the ratio of AdoMet decarboxylase activity to protein measured by either technique remained constant indicating that all of the increased activity occurring in response to DFMO was due to the increase in enzyme protein (Fig. 1). In contrast to the increased AdoMet decarboxylase content in prostates of rats treated with DFMO, no significant increase in the enzyme activity or protein occurred in the liver (Table I). This finding is not surprising in the light of the results for polyamine content of these tissues shown in Table 11. DFMO treatment produced a 54% reduction in putrescine and a 51% reduction in spermidine in the prostate but only a marginal effect on spermidine content in the liver. A somewhat greater reduction in prostatic polyamines could be achieved by supplementing the oral DFMO with intraperitoneal injection, and this treatment increased AdoMet decar- rats there was a coordinate increase in both enzyme activity and protein (Table I). As shown in Table 11, alone did not significantly reduce polyamine content at the time that AdoMet decarboxylase was increased. The proportionality between AdoMet decarboxylase protein and activity in the rat prostate clearly is demonstrated in Fig. 2 in which the results of a large number of measurements on individual animals treated with DFMO or for various periods of time are given. The activity is plotted on one axis and the protein on the other. It can be seen that activity is proportional to protein over a wide range (more than 30-fold). ylase by 12-fold. The combination of DFMO and also The amount of enzyme protein measured by RIA was slightly increased activity in both tissues but was less effective than greater than the amount measured by titration. This may alone. In both liver and prostate of the -treated indicate the presence of a small pool of inactive protein. In order to investigate the mechanism by which DFMO * Portions of this paper (including Experimental Procedures, part treatment increases prostatic AdoMet decarboxylase, rats of Results, and Figs. 1-5) are presented in miniprint at the end of were treated with cycloheximide to block protein synthesis this paper. Miniprint is easily read with the aid of a standard and the decline in enzytne activity and protein measured (Fig. magnifying glass. Full size photocopies are available from the Journal 3). The enzyme activity and amount of active protein meaof Biological Chemistry, 9650 Rockville Pike, Bethesda, MD sured by titration declined at the same rate which was signif- Request Document No. 85M-89, cite the authors, and include a check or money order for $5.20 per set of photocopies. Full size photocopies icantly reduced by DFMO treatment. The half-life in control are also included in the microfilm edition of the Journal that is rats was 35 min and in DFMO-treated rats it was 108 min. available from Waverly Press. The enzyme protein measured by radioimmunoassay declined boxylase activity and protein by 7-fold (Table I)., a competitive inhibitor of AdoMet decarboxylase, is known to increase the activity of the enzyme (12-14). As shown in Table I, treatment with 80 mg/kg doses of increased prostatic AdoMet decarboxylase activity and protein by about 25-fold and increased liver AdoMet decarbox- TABLE I Effect of DFMO and on liver and prostate AdoMet decarboxylase DFMO was administered as a 2% solution (ph 6) in drinking water and where indicated by intraperitoneal injection of 500 mg/kg doses every 12 h. Total exposure time to DFMO was 4 days. was given at a dose of 80 mg/kg 23 h before death. Results are shown as mean f S.E. for groups of 4 rats. AdoMet de- AdoMet decarboxylase Treatment Tissue protein carboxylase activity BY titration BY RIA pmollmg milliunitsjmg 85 f f f f f ND 430 f f f f f ND 600 f f 0.25 ND 1970 f f f f f 0.05 ND 860 f f f f f 0.04 ND TABLE I1 Effect of DFMO and on polyamine content Rats were treated with DFMO and as described in Table I. Control DFMO DFMO + Control DFMO DFMO + Putrescine c20 c20 52 f f f k f f 87 Polyamine content Spermidine Suermine nmoljg 602 f f f f f f f f f f f f k f f f 420

3 Regulation of S-Adenosylmethionine Decarboxylase ff10 PROTEIN (L,RIA;-,TITRATION) pmol/mg FIG. 2. Correlation of AdoMet decarboxylase activity with protein. extracts from rats treated with DFMO or to alter AdoMet decarboxylase activity were assayed for activity or AdoMet decarboxylase protein. Results are shown for protein determination by RIA (0) or titration (0). considerably more slowly with a half-life of 139 min in control rats and 213 min in the DFMO-treated animals. This suggests that the initial step in the degradation of AdoMet decarboxylase leads to loss of catalytic activity but does not result in the loss of immunoreactive protein. Analysis of the prostatic extracts by immunoblotting with the antiserum to AdoMet I I I I I decarboxylase also indicated that the loss of immunoactive protein was much slower than the loss of prostatic AdoMet MINUTES decarboxylase activity. Semiquantitative analysis by densito- FIG. 3. Effect of DFMO of half-life of AdoMet decarboxylmetric scanning of these immunoblots revealed only a 45% ase. Rats were pretreated with DFMO (2% in drinking water for 3 loss of the protein within 2 h in control rats and a 40% loss days; upper set of lines) or untreated (lower set of lines), and protein synthesis was blocked by administration of cycloheximide (20 mg/ in 3% h in the DFMO-treated animals. Also, there was no kg). The upper two sets of lines show results for prostate and the change in the molecular weight of the major band at 32,000 lower set for rat liver as indicated. AdoMet decarboxylase activity indicating that smaller fragments of the enzyme either did (m), protein by RIA (A), and protein by titration (0) were measured. not accumulate or were not recognized by the antiserum Results are given & S.E. for 4 estimations. (results not shown). The effect of cycloheximide treatment on liver AdoMet decarboxylase activity and immunoreactive protein was also by the more precise RIA technique (Fig. 3). However, prostatic tested (Fig. 3). The half-life for loss of AdoMet decarboxylase AdoMet decarboxylase activity was lost 3 times more rapidly activity was 47 min, and the protein was lost with a half-life than the protein (Fig. 3) indicating a significant tissue-specific of 67 min (Fig. 3). These results are in good agreement with difference. This may be due to a higher protease activity previous estimates of 50 and 65 min, respectively (13). Since directed against inactive proteins in the liver. Since the activ- DFMO did not increase AdoMet decarboxylase in liver, it was ity measurements and values for active AdoMet decarboxylase not possible to investigate the effect of spermidine depletion protein obtained by titration agree closely (Fig. 3) whereas on the half-life in this tissue. the RIA (Fig. 3) and immunoblots indicate a slower loss of DISCUSSION Although antisera to mammalian AdoMet decarboxylase immunoreactive protein and no change in its molecular weight, it appears that the initial step in the degradation of AdoMet decarboxylase in the prostate involves the loss of the have been described by several groups (13-16) the methods active site. This loss could, however, be brought about by used in this paper for investigating changes AdoMet in decar- proteolytic cleavage if only a small peptide is removed. This boxylase protein have significant advantages over previous is a reasonable possibility since the pyruvate prosthetic group work. The combination of the stoichiometric titration of the may be at the amino terminus (2,20). Many other mechanisms active site of AdoMet decarboxylase which measures active including a post-translational modification or selective deenzyme molecules and RIA which measures total immuno- struction of the pyruvate itself are also feasible. It is possible reactive protein provides a good opportunity to detect inactive that the accumulation of an inactive form of prostatic AdoMet forms of the enzyme. Earlier studies utilized immunotitration of activity with antisera of lower avidity (13, 14, 16), which is less accurate or sensitive than the present RIA. This work, which was carried out by immunotitration, revealed only a small difference in the rates of loss of AdoMet decarboxylase decarboxylase occurs only under the special conditions where protein synthesis is inhibited by cycloheximide, but this treatment should be useful for the isolation of this intermediate. Increased activity of AdoMet decarboxylase in response to DFMO has been observed in a variety of cultured cells (5, 6, activity and protein in rat liver after inhibition of protein 8, 21) and in some tissues of animals treated with this drug synthesis with cycloheximide (13). This finding is confirmed (21-24). It is very likely, although not proven conclusively,

4 9586 Regulation of S-Adenosylmethionine Decarboxylase that this increase is due to the decline in spermidine since I1 and Fig. 3). Therefore, itself rather than the altered addition of spermidine reverses the effect (5,6,8) and admin- polyamine content appears to retard degradation of AdoMet istration of spermidine reduces AdoMet decarboxylase activ- decarboxylase. Since it is a competitive inhibitor (10, 12) it ity (7, 25). This is also consistent with the lack of effect of may accomplish this by binding to the active site, but another DFMO on liver AdoMet decarboxylase (Table I) since DFMO possibility is that (which can be considered as a is relatively ineffective as an inhibitor of hepatic polyamine spermidine analog) displaces spermidine from some site biosynthesis (4, 21, 22) and did not deplete spermidine in our needed to bring about the initial inactivation. DFMO and experiments (Table 11). Our results indicate that all of the were not synergistic in increasing AdoMet decarboxincreased AdoMet decarboxylase activity in response to ylase in our experiments (Table I), but this combination DFMO is due to an increase in the amount of active enzyme produced clear signs of toxicity, and others using different protein and that a substantial part of the increase is caused protocols have seen additive effects (24). by an extended half-life of the enzyme. This conclusion is consistent with previous studies which only measured AdoMet REFERENCES decarboxylase activity (6, 8). In addition, our results indicate that spermidine levels affect predominantly the initial step in 1. Tabor, C. W., and Tabor, H. (1984) Annu. Rev. Biochem. 536, degradation which leads to the loss of enzyme activity. 2. Tabor, C. W., and Tabor, H. (1984) Adu. Enzymol. Relat. Areas The change in degradation rate is not sufficient to account Mol. Biol. 56, for all of the increase in AdoMet decarboxylase protein, and 3. Pegg, A. E. (1984) Cell Biochem. Funct. 2, some action on the rate of synthesis of the enzyme must also 4. Pegg, A. E., and McCann, P. P. (1982) Am. J. Physiol. 243, occur. The present antiserum should be valuable in studying c212-c221 the synthesis of AdoMet decarboxylase. The two faint bands 5. Pegg, A. E. (1984) Biochem. J. 224, Alhonen-Hongisto, L. (1980) Biochem. J. 190, of immunoreactive protein of higher molecular weight which 7. Hopkins, D., and Manchester, K. L. (1980) FEBS Lett. 109, were detected in some immunoblots may represent inactive precursors of the enzyme. (They are unlikely to be active 8. Mamont, P. S., Joder-Ohlenbusch, A.-M., Nussli, M., and Grove, since no corresponding labeled bands were produced when the J. (1981) Biochem. J. 196, active site was reacted with rnethyl-3h-decarboxylated 9. Pegg, A. E., Tang, K.-C., and Coward, J. K. (1982) Biochemistry AdoMet and sodium cyanoborohydride.) The presence of such a precursor or of a small pool of inactivated but not yet degraded protein could account for the slight difference in AdoMet decarboxylase protein measured by RIA and by titration (Fig. 2). Finally, it has already been shown that, a potent but reversible inhibitor of AdoMet decarboxylase (10, 12), leads to a large increase in the amount of AdoMet decarboxylase protein primarily by stabilizing the enzyme against degradation in vivo (2,3, 10, 12-14). Our results confirm this finding using the more precise RIA technique. It was suggested that the stabilization results from a reduced degradation of the enzyme protein when bound to the drug (reviewed in Refs. 2 and 12). The more recent findings that spermidine depletion results in an increase in AdoMet decarboxylase raise the possibility that, as suggested recently by Tabor and Tabor (2), the response to is mediated via reduction in spermidine since is an inhibitor of polyamine biosynthesis. However, the inhibitory effect of on polyamine synthesis in animals is quite transient and rapidly wears off as the drug is excreted AdoMet and decarboxylase is increased (10, 12, 26). As shown in Tables I and 11, brought about a 12- and 25-fold rise in AdoMet decarboxylase protein in rat liver and prostate, respectively, at a time when spermidine levels were not significantly different from controls. Hence its effect on AdoMet decarboxylase protein is not brought about via changes in spermidine content. does produce an increase in putrescine, and putrescine was reported to stabilize AdoMet decarboxylase in vitro (14, 27), but this is also unlikely to explain the increase in AdoMet decarboxylase protein since very high levels of putrescine in livers perfused with putrescine did not stabilize or increase AdoMet decarboxylase (28), and DFMO depletes putrescine but increases AdoMet decarboxylase and its half-life (Table 21, Porter, C. W., Dave, C., and Mihich, E. (1981) in Polyamines in Biology and Medicine (Morris, D. R., and Marton, L. J., eds) pp , Marcel Dekker, New York 11. Sjoerdsma, A., and Schechter, P. J. (1984) Clin. Pharmacol. Ther. 35, Williams-Ashman, H. G., and Pegg, A. E. (1981) in Polyamines in Biology and Medicine (Morris, D. R., and Marton, L. J., eds) pp , Marcel Dekker, New York 13. Pegg, A. E. (1979) J. Biol. Chem. 254, Sakai. T.. Hori.. C... Kano,. K.,. and Oka, T. (1979) Biochemistry 18,' Seyfried, C. E., Oleinik, 0. E., Degen, J. L., Resing, K., and Morris, D. R. (1982) Biochim. Biophys. Acta 716, Shain, S.'A., Hilliard, J. K., and de Leon, C. (1983) Endocrinology 113, Pankaskie, M., and Abdel-Monem, M. M. (1980) J. Pharm. Sci. 69, Markham,G. D., Tabor, C. W., and Tabor, H. (1982) J. Biol. Chem. 257, Shirahata, A., and Pegg, A. E. (1985) Fed. Proc. 44, Recsei, P. A., and Snell, E. E. (1984) Annu. Reu. Biochem. 53, Pegg, A. E., Poso, H., Shuttleworth, K., and Bennett, R. A. (1982) Biochem. J. 202, Wagner, J., Danzin, C., and Mamont, P. (1982) J. Chromatogr. 227, Danzin, C., Claverie, N., Wagner, J., Grove, J., and Koch-Weser, J. (1982) Biochem. J. 202, Kapyaho, K., Kallio, A., and Janne, J. (1984) Biochem. J. 219, "_ ". 25. Poso, H., and Pegg, A. E. (1981) Biochem. J. 200, Pegg, A. E. (1973) Biochem. J. 132, Poso, H., and Pegg, A. E. (1982) Biochemistry 21, Pegg, A. E., and Jefferson, L. S. (1974) FEBS Lett. 40, Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. (1971) J. Clin. Endocrinol. 33, Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, Bradford, M. M. (1976) Anal. Biochem. 72,

5 Regulation of 5'-Adenosylmethionine Decarboxylme 9587!:; 3, O.OO! ANTISERUM (pi x M.W

6 9588 Regulation of S-Adenosylmethionine Decarboxyluse " MINUTES