Regulation of polyamine biosynthesis by ... - Bioscience Reports

Bioscience Reports 5, 189-204 (1985) Printed in Great Britain

R e g u l a t i o n of p o l y a m i n e b i o s y n t h e s i s by a n t i z y m e and some r e c e n t d e v e l o p m e n t s r e l a t i n g t h e i n d u c t i o n of p o l y a m i n e b i o s y n t h e s i s to c e l l g r o w t h Review

E. S. CANELLAKIS, D. A. KYRIAKIDIS, C. A. RINEHART 3r., S.-C. HUANG, C. PANAGIOTIDIS and W.-F. FONG Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, U.S.A.

This review considers the role of antizyme, of amino acids and of p r o t e i n synthesis in the regulation of polyamine biosynthesis. The ornithine decarboxylase of eukaryotic ceils and of B s c h e r i c h i a c o l i can be non-competitively inhibited by proteins, termed antizymes, which are induced by diand polyamines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution. Evidence for the physiological role of the antizyme in m a m m a l i a n cells rests upon its identification in n o r m a l uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and a n t i z y m e in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein t h a t specifically binds to the a n t i z y m e of an o r n i t h i n e d e c a r b o x y l a s e - a n t i z y m e complex and liberates free ornithine decarboxylase from t h e complex. In B. c o l i , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by a r g i n i n e d e c a r b o x y l a s e , three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor l y s i n e decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins SI0/LI~ and L3~, respectively. The relationship of the a c i d i c a n t i z y m e to o t h e r known B. c o l i proteins remains to be determined.

Ig9

190

CANELLAKIS ET AL.

In mammalian ceils, ornithine decarboxylase can be i n d u c e d by a broad spectrum of compounds. These r a n g e from hormones and growth factors to natural amino acids such as asparagine and to non-metabolizable amino acid analogues such as a-amino-isobutyric acid. The amino acids that induce ornithine decarboxylase as well as those that promote polyamine uptake u t i l i z e the sodium d e p e n d e n t A and N t r a n s p o r t systems. C o n s e q u e n t l y , t h e y a c t in c o n c e r t and increase intracellular polyamine levels by both mechanisms. The induction of ornithine decarboxylase by growth factors, such as NGF, EGF, and PDGF as well as by insulin requires the presence of these same amino acids and does not occur in their absence. However, the inducing amino acid need not be incorporated into protein nor covalently modified. It is s u g g e s t e d t h a t the i n d u c t i o n of ornithine d e c a r b o x y l a s e activity and of polyamine biosynthesis occurs when the prerequisites for protein synthesis, cell division and growth are present. These include the availability of amino acids transported by Systems A and N and the a t t a i n m e n t of critical intracellular concentrations of these amino acids. Introduction

The r e g u l a t i o n of ornithine decarboxylase (L-ornithine carboxylyase, EC 4.l.1.17) and of polyamine biosynthesis has been reviewed (1-6). We shall discuss the regulation of polyamine biosynthesis by antizyme and the relationship of amino acids and protein synthesis to polyamine biosynthesis. The polyamines are among the few remaining metabolites about whose function we have very little solid information, even though they e x i s t in ceils in m i l l i m o l a r concentrations and their biosynthesis appears to be intimately related to cell division and growth. O r n i t h i n e d e c a r b o x y l a s e converts ornithine to putrescine and is widely distributed in eukaryotes and prokaryotes (7,8). Putrescine was f i r s t s h o w n to be an e s s e n t i a l g r o w t h f a c t o r in Eemophilus parainfluenzae by Herbst and Snell (9). Subsequently, H a m showed putrescine to be necessary for the optimal growth of several m a m malian cell lines (10) while Bottenstein and Sato ( l l ) and Pohjanpelto (12) i d e n t i f i e d putrescine a s the main factor responsible for the acceleration oi growth of human fibroblasts. Spermidine and spermine are formed by the addition of one and two aminopropyl groups, respectively, to the terminal amino groups of putrescine. In E s c h e r i c h i a c o l i , putrescine can also be formed through arginine decarboxylase (L-arginine carboxy-lyase, EC #.1.1.19), which decarboxylates arginine to form agmatine. The latter is then h y d r o l y z e d to p u t r e s c i n e by a g m a t i n e u r e o h y d r o l a s e ( a g m a t i n e amidinohydrolase, EC 3.5.3.11) (13,14) (Fig. 1). Mammalian

Cells

Beck et al. noted that administration of puromycin to rats induces liver ornithine decarboxylase (15). It was suggested that puromycin

POLYAMINE BIOSYNTHESIS

AND ANTIZYME

191

NH |

.H2

.H-c-~.2

(CHI25] CH -

[CH2)3

NH2

CH-

f

NH2

I COOH

COOH

Ornilhine...~

BIO DEGRADATIVE OOC

fArginine

~ BIO DEGRADATIVE ADC

BIOSYNTHETIC ODC ADC

~,.. ~ /

"-.~ ~,.-c-~>/ NH2

NH 2 Putrescine

Agmntine

.....----- S- AdenosylmetMonine

-'~s,Moc

NHz- (CHz) 4 - NH - [CH2) ~ - NH 2 Spermidine

Fig. l. Outline of the main metabolic reactions leading to the synthesis of the polyamines of E. co2i. ODC, ornithine decarboxylase; ADC, arginine decarboxylase; AUH, agmatine ureohydrolase; SAMDC, S-adenosylmethionine decarboxylase; Az, antizyme 1 and/or antizyme 2 and/or acidic antizyme. inhibited the synthesis of a protein inhibitor of ornithine decarboxylase (15). Fong et al. further pursued these studies in cell cultures and found t h a t H-35 h e p a t o m a cells exposed to putrescine produce a n o n - c o m p e t i t i v e i n h i b i t o r of ornithine decarboxylase that binds to ornithine decarboxylase in a salt-dissociable linkage (16). Since this n o n - c o m p e t i t i v e p r o t e i n i n h i b i t o r of ornithine decarboxylase was induced in response to the product of the reaction, it represented a novel mechanism of control of enzymatic activity, and we named it the antizyme of ornithine decarboxylase (17). Initially controversial, because of the difficulty in purifying the antizyme and because of the high c o n c e n t r a t i o n s of polyamines that were used at the time to induce the appearance of the antizyme, these observations have been confirmed (1g-35). Antizyme to ornithine decarboxylase has been induced in a variety of eukaryotic ceils including fibroblasts7 nerve and hepatic cells (19), as well as HeLa (26) and hepatoma cells (23,2#) and in thyroid ceils (2g) and in plant cells (32,33) as well as in tissue secretions such as milk ( 3 6 ) . The antizyme to ornithine decarboxylase was shown to exist also in normal uninduced cells ( l g ) . In such cells it was not found in the free form but in a bound form in the nuclei or in the post-ribosomal n u c l e o p r o t e i n p a r t i c l e s (the l a t t e r can be isolated by extensive

192

CANELLAKIS ET AL.

centrifugation oi the ribosome-free cell e x t r a c t s ) . High salt concentrations (0.3 M KCI) are required to extract the total antizyme of the post-ribosomal particles. The extracted material is of low specific activity. High specific activity antizyme can be extracted from these subcellular components by micromolar concentrations of putrescine and spermidine (18). It was suggested that the polyamines may interact in a specific manner with the antizyme, resulting in a dissociation of the antizyme from the ribonucleoprotein particles. In a d d i t i o n to p u t r e s c i n e , the n a t u r a l l y occurring poiyamines s p e r m i d i n e and s p e r m i n e also induce the a n t i z y m e to ornithine decarboxylase. Aliphatic diamines ranging from two to ten carbons in chain length have also been shown to induce the antizyme to ornithine decarboxylase (19,23,2~,2g,35,37). Consequently, it would appear that the inhibition of polyamine biosynthesis is a general cellular response when cells are exposed to aliphatic amines. The physiological role of the antizyme to ornithine decarboxyiase was investigated by Heller and Canellakis with starved cell cultures that have minimal levels of ornithine decarboxylase (19,34). In such cell cultures, free antizyme could be induced with concentrations of p u t r e s c i n e as low as 10-s to l0 -7 M, because such ceils contain minimal amounts of intracellular ornithine decarboxylase to complex the antizyme that is induced. Consequently, they are very sensitive indicators oi any free antizyme that may be produced. By alternately inducing ornithine decarboxylase (with asparagine) or its antizyme (with putrescine), and determining the activity of their respective free forms, it was shown that if the cells had been pre-induced for one activity, the appearance of the opposing activity was delayed. To induce measurable amounts of active ornithine decarboxylase in cells that had been pre-induced for antizyme it was necessary either to increase the concentration of asparagine or to extend the experiment for longer periods of t i m e . S i m i l a r l y , in ceils that had been p r e - i n d u c e d for o r n i t h i n e d e c a r b o x y l a s e , high c o n c e n t r a t i o n s of putrescine were required to induce free antizyme; alternatively, the cells had to be incubated ior extended periods at lower concentrations of putrescine. In agreement with the in vitro evidence that antizyme acted as a non-competitive inhibitor oi ornithine decarboxylase, at no time could the two activities be detected concurrently. These results p r o v i d e d e v i d e n c e for the i n v e r s e relationship between ornithine decarboxylase and its antizyme. More r e c e n t l y , the presence and the turnover of the ornithine decarboxylase-antizyme complex has been determined in vivo through t h e use oi d i i l u o r o m e t h y l o r n i t h i n e (DFMO) (38), an ornithine decarboxylase activated irreversible inhibitor of the enzyme. Hayashi and his collaborators (39) found that the DFMO-inactivated ornithine decarboxylase exchanges with the native ornithine decarboxylase that exists in an inactive complex with antizyme. This exchange results in t h e l i b e r a t i o n of the native ornithine decarboxylase which can be assayed enzymatically and permits quantitation of the inactive complex of o r n i t h i n e d e c a r b o x y l a s e and antizyme and of the rate of its disappearance. A similar assay has been used by Brosnan, who has s h o w n the e x i s t e n c e of such o r n i t h i n e d e c a r b o x y l a s e - a n t i z y m e complexes in milk (40) and independently by Mitchell, who has studied their kinetics oi apppearance in H-35 cells (41).


AND ANTIZYME

193

The physiological role of such a complex has to be considered. In the above experiments, Hayashi and Mitchell demonstrated that the ornithine decarboxylase-antizyme complex disappears rapidly following its formation in vivo. Since the disappearance of ornithine decarboxylase could only be incompletely accounted for by the appearance of the ornithine decarboxylase-antizyme complex, it is thought that the f o r m a t i o n of t h e s e c o m p l e x e s p r o v i d e s a more rapid means of degradation of ornithine decarboxylase (39,#1). However, these results do not exclude an alternate interpretation. We had earlier proposed that inactive complexes ol ornithine decarboxylase and antizyme become associated with subceIlular components to create reservoirs of an inactive or of a cryptic form of ornithine d e c a r b o x y l a s e from which a c t i v e ornithine decarboxylase can be r e l e a s e d rapidly upon demand (t,30). Arguments against such an interpretation would be that the total amount of cellular ornithine decarboxylase can be accounted for by the coincidence between the t i t r a t a b l e a c t i v e o r n i t h i n e d e c a r b o x y l a s e and the protein bound aH-DFMO (35). However~ most such studies have been made with partially purified cell extracts or i00 000 g supernatant fractions of cell extracts (35). Consequently, any forms of ornithine decarboxylase that are associated with subcellular fractions would not be measured. This limitation of these studies has been noted (35). The following evidence can be marshalled in favor of the alternate hypothesis. There exists in rat liver a protein that could be responsible for the a c t i v a t i o n o5 free ornithine decarboxylase from its i n a c t i v e complexes with antizyme. This is the anti-antizyme discovered by Hayashi and his collaborators (31,42743). This protein binds strongly to the antizyme present in an enzymatically inactive c o m p l e x of o r n i t h i n e d e c a r b o x y l a s e - a n t i z y m e complex, dissociates a n t i z y m e from its o r n i t h i n e d e c a r b o x y l a s e - a n t i z y m e complex and liberates active ornithine decarboxylase (31743). It should also be noted that upon exposure of ceils to aH-DFMO7 radioactivity is also associated with subcellular components (#4745)~ It has been assumed t h a t this r a d i o a c t i v i t y is due to the presence of active ornithine decarboxylase; however, Kyriakidis et al. found that 3H-DFMO reacts not only with active ornithine decarboxylase, but also with the inactive enzyme present in the ornithine decarboxylase-antizyme complex (46). Consequently, following t r e a t m e n t of cells with ~H-DFMO~ an ornithine decarboxylase-antizyme complex bound to subcellular components would be indistinguishable from active ornithine decarboxylase by autoradiography. Therefore, the possibility that the particle-bound radioactivity is due to o r n i t h i n e d e c a r b o x y i a s e - a n t i z y m e complexes should be seriously considered. As a non-competitive inhibitor of ornithine decarboxylase, antizyme has been used to measure amounts of ornithine decarboxylase and c o m p a r e t h e m with ornithine decarboxylase activity (35746). The quantitation of ornithine decarboxylase in cell extracts has also been performed with 3H-DFMO (47-#9) and with antibodies to ornithine d e c a r b o x y l a s e (35,#9-55). Under conditions of long-term induction (46-55) the increases in ornithine decarboxylase activity in mammalian c e l l s were a l w a y s a c c o m p a n i e d by corresponding increases in the amount of ornithine decarboxylase protein. It is b e c o m i n g a p p a r e n t t h a t when ornithine decarboxylase is

194

CANELLAKIS ET AL.

induced in tissues, the increase in the ornithine decarboxylase mRNA is much less than the increase in ornithine decarboxylase activity (56-59). This may be explained by an increased stability of the mRNA or by the possible existence of more than one species of ornithine decarboxylase. However, it has also been found by Kameji e t al. (59) t h a t upon i n d u c t i o n of ornithine decarboxylase with t h i o a c e t a m i d e , the total amount of free immunotitratable ornithine d e c a r b o x y l a s e p r o t e i n plus the mRNA associated immunotitratable newly synthesized proteins are also less than the amount necessary to account for the increase in enzyme activity. One factor that may help explain these discrepancies is the finding of Kitani and Fujisawa that the activity of the partially purified mammalian enzyme can be enhanced by phospholipids (60). It may also explain the rapid and transient activation that occurs within two minutes of exposure of mouse kidney slices to androgen, that was noted by Koenig et al. ( 6 1 , 6 2 ) , since t r o p h i c stimuli are known to activate phospholipid metabolism (63). The rat liver antizyme of ornithine decarboxylase has been recently purified 600 000-fold to homogeneity by Kitani and Fujisawa (64). This feat was only made possible following the comparably extensive purification of rat liver ornithine decarboxylase by Hayashi and his collaborators (65) and by Kitani and Fujisawa (66). The use of affinity columns of highly purified ornithine decarboxylase was a major tool in the purification procedure, resulting in a 5500-fold purification of rat liver antizyme in a single passage. The rat liver antizyme, moi.wt. = 22 000, and pI = 6.8, exhibits a high affinity for rat liver ornithine decarboxyiase, Keq = 1.4 x l0 n M-1. The maximal activity of antizyme that can be induced in rat liver by the administration of p u t r e s c i n e is a p p r o x i m a t e l y equivalent to the maximal amount of ornithine decarboxylase that can be induced following administration of dexamethasone (29,64). The question of the existence of alternate forms of antizyme and of ornithine decarboxylase has been raised (1,30) and remains to be f u r t h e r elucidated. Antizyme preparations do not completely inactivate ornithine decarboxylase activity (46) and there is evidence that there are components in tissue extracts that modify the interaction between ornithine d e c a r b o x y l a s e and a n t i z y m e ( 2 1 ) . Chromatographic separation of two forms of antizyme from chicken liver has been achieved by GrilIo et al. (67). Similarly, there is a great deal of cumulative evidence for the existence of more than one form of ornithine decarboxylase (68-71), and recently Berger et al. (57) have identified two distinct mRNAs in mouse kidney coding for ornithine decarboxylase. The r o l e of biosynthesis

amino

acids

in

the

regulation

of

polyamine

Chen and Canellakis found that ornithine decarboxylase could be induced in a salts-glucose solution (Earle's Balanced Salts Solution) and have used this solution to determine the critical requirements for polyamine biosynthesis (52). They found tha% contrary to what had been found in more complex incubation media, cAMP would not induce ornithine decarboxylase unless a minimal concentration of asparagine,

POLYAMINE BIOSYNTHESIS AND ANTIZYME

195

which in itself was inadequate to induce ornithine decarboxylase, was present (52). It became apparent that the e f f e c t of cAMP was indirect and was mediated through asparagine. Using the salts-glucose media, Costa et al. found asparagine to be a r e q u i r e m e n t f or t h e i n d u c t i o n of o r n i t h i n e decarboxyl ase by luteinizing hormone in CHO ceils (54), while Viceps-Madore et al. (5.5) s h o w e d t h e i n d u c t i o n of ornithine decarboxylase to be Na + dependent and t hat in addition to asparagine, which was the best i n d u c e r , c e r t a i n n o n - m e t a b o l i z a b l e amino acids such as c~-aminoisobutyric acid9 N-methyl asparagine and others were also e f f e c t i v e inducers of ornithine decarboxylase. These results established t hat the metabolism of the inducing amino acid and its covalent modification was not necessary for the induction of ornithine decarboxylase (55). Rinehart and Chen showed that the natural amino acids, as well as n o n - m e t a b o l i z a b l e a n a l o g u e s , that utilize the System A transport system stimulate the uptake of polyamines (72). It has now become evident that the sodium dependent System A and N amino acid transport systems play a central role in polyamine biosynthesis. Amino acids t hat are transported by these systems not only stimulate the uptake of polyamines but are also required for the induction of ornithine decarboxylase act i vi t y (73). However, it was also found t h a t in order t hat these amino acids induce ornithine decarboxylase, it is necessary that they achieve a critical intracellular concentration. Furthermore, much as cAMP would not induce ornithine decarboxylase in the absence of asparagine, it was now found that growth factors such as plate/et derived growth fact or, nerve growth factor, epidermal growth f a c t o r and insulin, added to a variety of r e s p o n s i v e c e l l s , will not induce ornithine decarboxylase unless a System A or N inducing amino acid, such as asparagine, is present (7#). S i g n i f i c a n t l y , a s p a r a g i n e c o u l d agai n be replaced by an unnatural amino acid analogue such as c~-amino-isobutyric acid. It was c o n c l u d e d t h a t growth fact ors do not induce ornithine decarboxylase act i vi t y directly, but their e f f e c t is mediated through S y s t e m A and N a m i n o acids and does not require the covalent modification of these amino acids. It is evident t hat the induction of o r n i t h i n e d e c a r b o x y l a s e a c t i v i t y by cA MP and by cAMP-mediated growth f ac t or s (NGF, EGF and PDGF) as well as by insulin is not a s e l f - c o n t a i n e d r e a c t i o n but is dependent upon the presence of an inducing amino acid. Since c~-amino-isobutyric acid is known not to be metabolized intracellularly (75976), the inducing amino acids may be acting as gratuitous inducers, in the manner of isopropyl 8-D-thiogalactose-pyranoside (IPTG) during the induction of g-galactosidase in E. coli

(77).

Consequently, these two coordinated e f f e c t s of the System A and N amino acids upon the uptake of polyamines and upon the induction of ornithine decarboxylase, reinforce each other and both contribute to an increased intraceJlular polyamine concent rat i on. E. coli

Kyriakidis et al. (20) isolated from E. c o l i a non-competitive inhibitor protein of the E. c o l i ornithine decarboxylase. This protein was purified to homogeneity, and found to be an acidic protein, pI = 3.8, and to have an apparent mol.wt. = #9 500. Its intracellular

196

CANELLAKIS ET AL.

concentration increased when E. c o l i were grown in the presence of i n c r e a s i n g concentrations of putrescine and spermidine and it inhibited the mammalian ornithine decarboxylase. Because of the overlap in these various properties with the mammalian antizymel this inhibitory protein o f E. c o l i was also named antizyme (20,78,79). Two basic proteins, with pI greater than 9.51 have since been purified from E . c o l i , with biological properties very similar to those found for t h e a c i d i c a n t i z y m e l t h e s e were named, provisionally1 antizymes 1 and 2 (Az-I and Az-2) (78). The two basic E. e o l i proteins were shown to inhibit both the b i o s y n t h e t i c o r n i t h i n e d e c a r b o x y l a s e and the biosynthetic arzinine decarboxylase (Fig. t); they did not inhibit the corresponding biodegradative ornithine, arginine and lysine decarboxylases of E. c o l i nor did they inhibit S-adenosyl methionine decarboxylase (79). Unlike the biosynthetic decarboxylasesl the biodegradative decarboxylases are not constitutive, but are induced when E. c o l i are grown in low pH medial their function appears to be one of raising the intracellular pH (S0-82). To define more closely the specificity of inhibition of decarboxylases by the various antizymes, a lysine decarboxylase that requires pyridoxal phosphate and does not decarboxylate ornithine or arginine was p u r i f i e d about 200-fold from E. c o l i . It differs from the well-studied biodegradative lysine decarboxylase of E. c o l i which has a pH optimum of 5.5 (83), in having a high pH optimum, about 9.0 (C. Panagiotidis, unpublished results). It appears to be a biosynthetic enzyme similar to the biosynthetic ornithine decarboxylase and arginine decarboxylases (8218#) and is reminiscent of the lysine decarboxylase that was referred to by Leifer and Maas (85). However, cadaverine, the product of lysine decarboxylase activityl is known not to replace putrescine for growth (86). This new lysine decarboxytase was also found not to be inhibited by these two basic antizyme proteins (C. Panagiotidis, unpublished results). Both the acidic antizyme of E. c o l i and the rat liver antizyme were also tested for their ability to inhibit these various decarboxylases and the results are presented in Table I. It is evident that all these proteins that were first identified as non-competitive inhibitors of o r n i t h i n e d e c a r b o x y l a s e inhibit only the polyamine biosynthetic decarboxylases. Both the acidic and the basic E . c o l i antizymes inhibit the structurally dissimilar rat liver ornithine decarboxylase. In a complementary fashionl the rat liver antizyme inhibits the biosynthetic ornithine and arginine decarboxylases o f E . coli~ in fact, it appears to be more effective against the E. c o l i ornithine decarboxylase than the rat liver enzyme. Huang et al. (87) found that the inhibition of E. c o l i ornithine decarboxylase caused by the acidic antizyme and by the basic E. c o l i antizymes is relieved to different degrees by DNA and by synthetic nucleic acid polymers indicating preferences for certain nucleic acid sequences (87). The inhibition of rat liver ornithine decarboxylase by the E. c o l i antizymes shown in Table 1 is relieved by nucleic acids (S. C. Huang, unpublished results). It should be noted, however, that t h e i n h i b i t i o n s e x e r t e d by rat liver antizyme upon these various decarboxylases i s not prevented by nucleic acids (W. Fongl unpublished results).


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197

Table I. Comparison of activity of various antizyme preparations against various decarboxylases In each case, 2-3 units of the decarboxylase were titrated with the antizyme fraction indicated. The units of antizyme required to lower the decarboxylase activity by 20% was determined, as estimated from the linear portion of the curve. Decarboxylases Antizymes

E. coli ODC

E. coli ADC

Rat liver ODC

Antizyme units required to inhibit 20 decarboxylase units E. coli

S20/L26 L34 Acidic Az

20* 20* 20*

70 64 133

188 172 182

48

20*

Rat liver

Antizyme

II

ODC = ornithine decarboxylase; ADC = arginine decarboxylases; * Inhibitor units in these cases were defined as being equivalent to the 20 decarboxylase units (see above). All other inhibitor values were normalized to this value.

It was iound that a variety of synthetic nucleic acid polymers, including ribo- and deoxyribo- polymers, will also relieve the inhibition oi ornithine decarboxytase by the E. a o l i antizyrnes (87); additional experimental data in support of this s t a t e m e n t are presented in Figs. 2 and 3. These results and the specificities indicated are not unlike those obtained for various DNA and other nucleic acid binding proteins which, in a d d i t i o n to their indicated specificity for speciiic DNA sequences, also exhibit varying degrees of specificity for binding to a variety of synthetic nucleic acid polymers (g8-91). The ability of DNA and other nucleic acids to relieve the inhibition of ornithine decarboxylase by the acidic antizyme (pI = 3.8), and the relative specificity shown in the binding of these proteins to various nucleic acid sequences, suggests that this reaction may prove to be more s p e c i f i c for certain nucleotide sequences and may not only represent a non-specific interaction between basic proteins and acidic nucleic acid polymers. At this time additional experiments are under way in our laboratory to understand the exact mode of this interaction and its underlying specificity. Panagiotidis and Canellakis (92) determined the partial amino acid sequences of Az-1 and of Az-2 and found them to be identical with c o r r e s p o n d i n g amino acid sequences of the $20/L26 (93) and L3~ r-proteins (94) respectively, within the limits of the methods used. The identity of Az-I and of Az-2 with the respective r-proteins was f u r t h e r v e r i f i e d by i s o l a t i n g ribosomes from E. c o l i MA255 by standard procedures (95). The basic ribosomal proteins were then

198

CANELLAKIS ET AL.

..f." 2.0

Dolv

f dA) ~-'~"

~-

~

p o ~ y (dI

o

/ H Z

o

y/

/ H

/

/

. ,

p o l y (dA)

i.{

0

i

0

i

50

J

i00

D~0•

150

HOMO~LY~RS

i

200

(nz)

Fig. 2. Reversal of the inhibition of ornithine decarboxylase by antizyme i, through the addition of deoxynucleotide homopolymers. E. coli ornithine decarboxylase (2.0 units) was inhibited with 1.8 units of E. c o l i antizyme 1 and the resultant complex was titrated with deoxynucleotide homopolymers.

extracted (96). Partial purification of these basic ribosomal proteins through a CM-Biogel A column9 followed by electrophoresis of this fraction in an acid-urea acrylamide gel9 showed two main components which t r a v e l l e d to the s a m e positions as did A z - I and Az-2. Furthermore9 their inhibition of ornithine decarboxylase activity was found to be relieved by added DNA. These experiments established that Az-1 is in fact the S20/L2s r-protein while Az-2 is the L34 r-protein. The $20/L26 is a basic inter-ribosom.al.protein that is shared by the s m a l I and large ribosomal subunits (93), while the L34 r - p r o t e i n is a basic protein associated exclusively with the large ribosomal subunit (94). Holtta et al. had found that the activity of the biosynthetic B. coli o r n i t h i n e d e c a r b o x y l a s e is greatly increased by nucleotides, especially GTP (97)~ while it is inhibited by ppGpp (98). These investigators considered the requirement of GTP for protein synthesis and the accumulation of ppGpp during starvation of E. c o l i that are under stringent control9 and suggested that changes in polyamine pools may be responsible for the stringent e f f e c t in E. c o l i (98). The accumulation of ppGpp leads to the cessation of stable RNA synthesis (999100) and appears to be related to the fidelity of protein synthesis (1019102) .


Z

2.0

0/ ,/

u O

I99

1.0

r(

i

0

i00

i

200 RIBONUCLEOTIDE

i

i

300 HOMOPOLYMERS

400 (ng)

Fig. 3. Reversal of the inhibition of E. coli ornithine decarboxylase by antizyme I, through the addition of r i h o n u c l e o t i d e homopolymers. Experimental details as in the legend to Figure 2, e x c e p t that the titration was performed with ribonucleotide homopolymers, rA, rG, rC and rU refer to poly A, poly G, poly C and poly U respectively.

Sakai and Cohen examined the suggestion of Holtta et al. in a K+ requiring E. coli mutant (I03) maintained at various growth rates. They found a lack of correlation between the putrescine levels, the levels of GTP or of ppGpp and the rates of RNA synthesis. H o w e v e r , the c o n t r o l mechanism may be more complex than originally assumed, and it may be necessary to reconsider the Holtta et at. suggestion (98). It may be necessary to take into account the i n h i b i t i o n of o r n i t h i n e decarboxyiase by the $20/L26 and the L3~ r - p r o t e i n s because, d u r i n g s t a r v a t i o n of bacteria that are under s t r i n g e n t control, the synthesis of ornithine decarboxylase and the uptake of polyamines are also inhibited (10#). Furthermore, recent e v i d e n c e suggests t h a t there may exist a closer interrelationship between p o l y a m i n e s and p r o t e i n s y n t h e s i s . In addition to the well-known stimulation of the rate of protein synthesis in vitro by p o l y a m i n e s , Cooper et ai. have found that hypusine, a putrescine derivative, is covalently linked to the protein elongation factor E f - l (105). In a d d i t i o n , M i t s u i e t a / . (106) have shown that, in a polyamine requiring strain of E. c o l ] , the addition of polyamines is necessary for the optimal synthesis of a discrete 62K protein. One function of this 62K protein is to stimulate the synthesis of MS2 phage RNA replicase in the presence of sperm]dine (106).

200

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Discussion

The E . c o l i ornithine decarboxylase is known to be inhibited by a variety of basic and acidic proteins. These include polylysine (87), histones and heparin (S.C. Huang, unpublished e x p e r i m e n t s ) . The rat liver ornithine decarboxylase has been shown to be inhibited by the acidic heparin (60). However, it should be noted that the relatively n e u t r a l (pI = 6.8) r a t liver antizyme inhibits both the E . c o l i biosynthetic ornithine and arginine decarboxylases and does not inhibit the corresponding E. coil biodegradative decarboxylases nor lysine decarboxylase. On the other hand, the acidic E . c o l i ant i zym e and the basic S=0/L26 and Ls~ r-proteins inhibit the biosynthetic E . c o l i ornithine and arginine decarboxylases as well as the rat liver ornithine d e c a r b o x y l a s e but not t h e c o r r e s p o n d i n g biodegradative E . c o l i e n z y m e s nor t h e E . c o l i lysine d e c a r b o x y l a s e . There appears t h e r e f o r e to be an underlying specificity of interaction among these biosynthetic polyamine enzymes with certain proteins which is masked by an apparent non-specificity in their interaction with various basic and acidic polymers. Another underlying similarity in the behavior of the E . c o l i and the rat liver polyamine biosynthetic enzymes is that their inhibition by these various inhibitory proteins can be relieved by other polymers. On t h e one hand, t h e i n h i b i t i o n of both the rat liver ornithine decarboxylase and of the E . c o l i ornithine and arginine decarboxylases by the E . c o l i inhibitory proteins is reversed by nucleic acids. On the other hand the inhibition of the rat liver ornithine decarboxylase by rat liver antizyme is overcome only by a protein and not b y nucleic acids. It is as yet unclear whether the acidic antizyme and the 520/L26 and the L3~ r-proteins are the only acidic and basic E . o o l i proteins that inhibit the polyamine biosynthetic decarboxylases. Our method of isolation of these proteins includes acid ext ract i on of the 10 000 g supernatant fraction o f E . e o l i lysates (78). During this procedure t h e only e f f e c t i v e i n h i b i t o r s of ornithine decarboxylase that are e x t r a c t e d a r e the acidic antizyme and the basic $20/L2~ and L34 r-proteins. It is t h e r e f o r e possible that these enzymes are inhibited by other acidic or basic proteins o f E . c o l i which our method does not extract. The interaction between the biosynthetic polyamine enzymes and the ribosomal proteins suggests that it may r e f l e c t upon the rates: of protein synthesis and/or upon the rates of polyamine synthesis in E. coll. The interrelationship between polyamine synthesizing enzymes and protein synthesis is further a c c e n t u a t e d by the finding that an increase of the intracellular concentration of cert ai n amino acids is in i t s e l f a d e q u a t e f or t h e i n d u c t i o n of p o l y a m i n e b i o s y n t h e s i s in eukaryotic ceils. Furthermore, growth factors and hormones will not induce polyamine biosynthesis in these cells unless the inducing amino acids are present and unless the intracellular concentration is above a minimal concentration range. These data suggest that the presence of critical concentrations of amino acids is necessary for the initiation of polyamine-dependent growth processes. Should o t h e r such inhibitory proteins to polyamine synthesizing enzymes exist, it will be most interesting to define their physiological


201

function and to det er m i ne whether their function is dependent upon th e v a r i o u s p o l y a m i n e s y n t h e s i z i n g e n z y m e s . It remains to be d e t e r m i n e d w h e t h e r this i nt e r pl ay between polyamine synthesizing enzyme, nucleic acid associated proteins, nucleic acids and amino acids has a physiological function, such as permitting the modulation of p o l y a m i n e biosynthesis at localized regions of RNA and DNA and modifying nucleic acid dependent reactions, or whether we are noting reactions with limited physiological significance.

Acknowledgements Supported by a USPH Service Grant CA 26546, by a Research Training Grant CA 09085 and by a Research Career Award GM 03070 to E.S.C.

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