Biochimica et Biophvsica Acta 873 (1986) 53-61 53 Elsevier

BBA 32577

Effec t of N-alkyl and C-alkylputrescines on the activity of orni th ine

decarboxylase f rom rat liver and E. coli *

Oscar Ruiz, Danie l O. A lonso -Gar r ido , Grac ie la Bulda in

and Rosa l ia B. F r y d m a n

Facultad de Farrnacia y Bioqu~mica, Universidad de Buenos Aires, Jun~n 956 Buenos Aires (Argentina)

(Received March 17th, 1986)

Key words: Polyamine: Putrescine; N-Alkylputrescine; C-Alkylputrescine; Ornithine decarboxylase; (Rat liver, E. coli)

N-Methyl-, N-ethyl-, N-propyi-, N-butyl-, N,N-dimethyl- and N,N'-dimethylputrescines were assayed as inhibitors of ornithine decarboxylase (EC 4.1.1.17) from rat liver and from Escherichia coli. They were found to be poor inhibitors, with the exception of N-propylputrescine and N,N-dimethylputrescine, which were inhibitory at 25 mM. A homologous series of 1-alkylputrescines ranging from l-methylputrescine (l,4-diaminopentane) to 1-heptylputrescine (l,4-diaminoundecane) was assayed for effect on the activity of ornithine decarboxylase from the same sources, l-Methylputrescine (5 mM) inhibited the mammalian enzyme, while the higher homoiogues showed significantly less inhibitory activity. When assayed on the bacterial enzyme, l-methylputrescine (5 mM) was not inhibitor, while the higher homologues showed inhibitory effects. At higher concentrations, 1-methylputrescine and l-heptylputrescine were the best inhibitors of these series of rat liver ornithine decarboxylase. When l-methylputrescine, 2-methylputrescine, 1,2-dimethyiputrescine, 1,3-dimethylputrescine and 1,4-dimethylputrescine were assayed as inhibitors of the decarboxylase, 2-methylputrescine was found to be the best inhibitor of the rat liver enzyme, while 1,3-dimethylputrescine was the best inhibitor of the bacterial enzyme. 1,4-Dimethylputrescine (2,5-di- aminohexane) did not inhibit the enzyme from either source. Both, 2-methylputrescine and l-methylputres- cine, as well as the 1,2- and 1,3-dimethylputrescines were competitive inhibitors of the enzyme, and a K i of I mM was obtained for 2-methylputrescine when the rat liver decarboxylase was used. N-Methyl, 1-methyl and 2-methylputrescines were found to inhibit in vivo the activity of rat liver ornithine decabroxylase which had been previously induced by thioacetamide treatment. 2-Methylputrescine (50/~mol/100 g body weight) was found to be the best in vivo inhibitor (93% inhibition), while putrescine under similar conditions inhibited 56% of the enzymatic activity.

Introduction

The essential role of polyamines in growth and cellular division is well documented [1 3]. The first metabolite of the polyamine biosynthetic pathway is putrescine (1,4-diaminobutane), which

* Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday.

is formed by the decarboxylation of ornithine catalyzed by the pyridoxal-phosphate dependent enzyme L-ornithine decarboxylase (L-ornithine carboxylase, EC.1.1.17) [4]. Putrescine is amino- propylated to give spermidine (N-(3-aminoprop- yl)putrescine), which is again aminopropylated to give spermine (N,N'-di(3-aminopropyl)putres- cine). The sequential action of two decarboxylases and two aminopropyltransferases is involved in

0167-4838/86/$03,50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

54

these metabolic transformations. Polyamine bio- synthesis is a finely modulated process which is now fairly well documented [5,6]. Since poly- amines were shown to be involved in many im- portant biological processes such as DNA repli- cation, RNA synthesis, translation, transport and membrane reactions, the potential relevance of inhibiting the ornithine decarboxylase activity as a way to affect cell replication was the aim of much research effort. Many synthetic analogues of ornithine as well as a number of synthetic poly- amines were assayed as inhibitors of ornithine decarboxylase activity both in vitro and in vivo. One of the derivatives, 2-difluoromethylornithine, was found to be a powerful irreversible inhibitor of ornithine decarboxylase of eucaryotes, and 2- methyl-4-ethynylputrescine is very likely the most powerful irreversible inhibitor of this enzyme [7]. 1,4-Diaminobutene and 1,4-diaminobutyne were shown to be potent competitive inhibitors of rat liver ornithine decarboxylase [8].

Several attempts have been made to correlate polyamine structure and its capacity to influence the biological processes. Morris et al. [9-11] synthesized spermidine analogues and tested their action on cell growth and macromolecular synthe- sis. They changed the distance between the nitro- gen atoms by altering the number of methylene groups between them. Canellakis and coworkers [12] examined the effect of the commercially avail- able a,w-diaminoalkanes with 3 to 12 carbon atoms on the induction of ornithine decarboxylase and its antienzyme, looking for an inhibitory ef- fect on the biosynthesis of polyamines from its precursors. Similar studies using a,,0-diaminoal- kanes were also performed by other workers [13-16]. The effect of several N-alkylputrescines on the growth and in vivo protein synthesis of E. coil (BGA-8 strain) was also examined [17].

In this report we study the effect of a number of N-alkylputrescines and C-alkylputrescines which were recently obtained by synthesis [18,19] on the activity of ornithine decarboxylase in the search for a correlation between the substitution pattern of the 1,4-diaminobutane skeleton and the inhibitory effect on ornithine decarboxylase activ- ity. The N-alkylputrescine derivatives were the N-methyl-, N-ethyl-, N-propyl-, N-butyl-, N,N'- dimethyl- and N,N-dimethylputrescines (Fig. 1).

H ~ p ~ N H R 2'"

R-CH 3 ; C2H 5 ; n-C3H 7 n-C4Hg

N -AlkyEput rescines

N(CH B ) . . . . ~ " NHCH3 H2 N / ~ ~ L. PiBPir~l

N, N- D, methylputrescine N, N ' - Dimet hylputr'esctne

H 2 N ~ NH2 R=CH 3 ;C2H 5 ; n-C3H7 ; n -C4H 9 ; n -C5H l l ; n-C6H13 ; n-C7H15

I - Alkylputrescines

CH 3 CH3 / J - ~ - / N H 2 ~ ~ , / ~ - - J " - ~ "NH2

H2 N / v v "2

2 - Methyiput rescine 1, 2 - Dimethylput resclne

CH 3 CH 3 CH3 CH 3 H2 N . / ' - - ~ j ~ . ~ / N H 2 H 2 N / A " - . . . / ~ . . ~ NH2

1,3 - DirnethylDutrescine 1,4 -Dirnethylputresctne

Fig. 1. Chemical structures of the N-alkylputrescines and C-alkylputrescines used in this study.

The C-aikyl derivatives were 1-methyl-, 1-ethyl-, 1-propyl-, 1-butyl-, 1-pentyl-, 1-hexyl-, 1-heptyl-, 2-methyl-, 1,2-dimethyl-, 1,3-dimethyl- and 1,4-di- methylputrescines (Fig. 1).

Materials and Methods

Materials The N-alkylputrescines and the 1-alkylputres-

cines were obtained by synthesis [18,19]. N, N-Di- methyl- and N,N'-dimethylputrescines were ob- tained as described elsewhere [17]. 2-Methyl- putrescine, 1,2-dimethyl-, 1,3-dimethyl- and 1,4- dimethylputrescines were obtained from the corre- sponding pyrroles by ring-opening with hydroxyl- amine and reduction of the dioximes following the described procedures [19]. All the diamines were obtained as their dihydrochlorides and their purity was checked by TLC and by derivatization to the corresponding bis(benzyloxycarbonyl) derivatives. L-[1-14C]Ornithine (specific radioactivity 52-59 mCi/nmol) and Protosol were obtained from New England Nuclear Corp. (Boston, MA). Putrescine dihydrochloride, spermidine trihydrochloride, pyridoxal phosphate, dithiothreitol, phenylmethyl- sulphonyl fluoride (PMSF), poly(ethylene glycol) (M,. 15000) and EDTA were purchased from

Sigma Chemical Co. All other reagents were of analytical grade. E. coli MRE 600 was a gift from Dr. S.H. Goldemberg (Instituto de Investigaciones Bioquimicas, Buenos Aires). Female Wistar rats (180-220 g) were used.

Methods Animal treatment. Thioacetamide and all the

other chemicals were administered to the rats by intraperitoneal injection of a neutral solution in 0.9% sodium chloride. The concentrations were adjusted so that the volume injected did not ex- ceed 0.7 ml. The hydrochlorides of the amines were adjusted to neutral pH with a sodium hy- droxide solution and the solutions were used im- mediately.

Ornithine decarboxylase was induced in the rat liver by administration of 100 mg of thioaceta- mide per kg body weight 18-20 h before they were killed. When the in vivo effect of the synthetic diamines and putrescine was assayed, the rats were treated with thioacetamide(10 mg/100 g body weight) for 15 h, after which 50 t~mol/100 g body weight were injected intraperitoneally and the rats were killed 3 h later. The controls were injected with saline solution. The livers were excised and ornithine decarboxylase was prepared as described below,

Preparation of rat liuer and E. coli ornithine decarboxylase. The enzyme was prepared from the livers of thioacetamide-treated rats by homogeni- zation in 3 vol. 50 mM potassium phosphate buffer (pH 7.4) containing 5 mM NaF, 0.1% poly(ethyl- ene glycol), 0.1 mM EDTA, 1 mM PMSF and 1 mM dithiothreitol, using a Potter-Elvehjem glass homogenizer. The homogenate was centrifuged at 20000 x g for 20 rain and the supernatant was centrifuged again at 150000 x g for 60 min. The supernatant was partially purified by ammonium sulphate precipitation, the active fraction which precipitated between 25 and 55% ammonium sulphate was dissolved in the dialysis buffer (20 mM Tris-HCl buffer (pH 7.4)/1 mM EDTA/0.1% poly(ethylene glycol)/1 mM PMSF/10 mM mercaptoethanol) and was dialyzed overnight against 3 1 of the same buffer. The enzyme main- tained about 90% of its original activity and its specific activity was 0.08-0.12 nmol 14CO2 re- leased/min per mg protein. Further purification

55

was obtained by precipitation of the enzyme at its isoelectric point, followed by resuspension of the latter in the buffer used for homogenization of the livers. The specific activity of this preparation was 4.22 nmol ~4CO2 min per mg protein. This puri- fied preparation was very unstable and most of the experiments were performed with the amonium sulphate partially purified enzyme.

E. coli cells (0.5 g wet weight) were resuspended in 5 ml of 10 mM Tris-HC1 buffer (pH 7.4) containing 0.1 mM EDTA and 1 mM dithiothrei- tol, and the suspension was sonicated in an Ultra- sonic Inc. disintegrator at a power setting of 4 for 5 min (at intervals of 30 s). The suspension was centrifuged at 20000 x g for 20 min and the su- pernatant was then centrifuged again at 150 000 x g for 90 min. The clear supernatant was used for measurements of ornithine decarboxylase activity.

Assay of ornithine decarboxylase actiuity. The standard incubation mixture contained in a final volume of 120 /~1:50 mM Tris-HC1 buffer (pH 7.4), 0.1 mM EDTA, 5 mM NaF, 0.2% poly(ethyl- ene glycol), 4 mM dithiothreitol, 4 mM pyridoxal phoshate and 50 ffl enzyme (0.3 mg protein). The reaction was initiated by the addition of 0.6 mM L-[1-14C]ornithine (65 000 dpm). Incubations were run for 40 min at 37°C with constant shaking. The reaction was stopped by addition of 100 ffl of 4 M citric acid, and the incubation was continued for an additional 40 min with constant shaking. The ~4CO2 released was trapped by 50 ffl Protosol adsorbed on Whatman 1 filter strips suspended above the reaction tubes in glass wells identical to the plastic ones of Kontes. The paper strips were then placed in 10 ml of Omnifluor Toluene (New England Nuclear) and radioactivity was de- termined in a Liquid Scintillation counter. When the biosynthetic ornithine decarboxylase of E. coli was assayed, essentially the same incubation mix- ture was used except for the buffer, which was 10 mM Hepes (pH 8.2), and ornithine concentration, which was 5.7 mM (65 000 dpm). Incubation con- ditions and the assay for 14C02 were those de- scribed above.

Protein determinations. Proteins were measured using the methods of Lowry et al. [20] and Brad- ford [21]. When the former method was used the samples were precipitated with 20% w / v trichloro- acetic acid and the precipitate was dissolved in 0.1

5 6

M sodium hydroxide, to prevent interference in the assay by the mercaptoethanol used [22].

In every experiment ornithine decarboxylase activity was the mean of at least three separate estimations of protein on separate samples that agreed within 10%.

Polyamine determinations. Polyamine content of liver extracts was determined by addition of 0.2 M perchloric acid followed by dansylation of the supernatants according to Dion and Herbst [23] and TLC analysis of the dansyl derivatives [24].

Results

Effect of N-alkylputrescines on the activity of ornithine decarboxylase from rat liver and E. coli

Putrescine is a known competit ive inhibitor of ornithine decarboxylase from eukaryotes and pro- karyotes [1-3]. A number of N-alkylputrescines (Fig. 1) were assayed in order to examine their possible inhibitory activities on ornithine de- carboxylase from both sources. At the concentra- tions used (5 mM), putrescine was found to inhibit 28% of the activity of rat liver decarboxylase and 19% of the activity of ornithine decarboxylase from E. co#. Of the N-alkylputrescines assayed (N-methyl- , N-ethyl-, N-propyl-, N-butyl, N,N- dimethyl- and N,N'-dimethylputresc ine) only N- propylputrescine inhibited (18%) the mammalian decarboxylase, while the other putrescine deriva- tives did not affect its activity at the concentra- tions used. In the case of the bacterial enzyme, all the N-alkylputrescines assayed were slightly in- hibitory (10-13%), while N-propylputrescine was found to be a better inhibitor (27%) than putres- cine itself. The effect of N-propylputrescine could be due to its structural similarity with spermidine, which is known to be a poor inhibitor of the mammalian enzyme and a good inhibitor (better than putrescine) of the bacterial enzyme [25].

Inc reas ing c o n c e n t r a t i o n s of the N-al - kylputrescines resulted in inhibitions of the rat liver decarboxylase (Fig. 2). N-methyl-, N-ethyl-, N-butyl- and N,N-dimethylputrescine showed an inhibition of 10 15% at concentrat ions of 25 mM. N, N-dimethylputrescine did not show any inhibi- tory effect up to a 8 mM concentrat ion and in- hibited 60% of the enzymatic activity at a 25 mM concentration. N-propylputrescine inhibited 70%

~ ". '~ N, N' Dimethylputrescine

",r \ ,

l ' . . \ \ ' ,

30 N Propylput resci

l o

0 I i g I I I I i I i , i i I i ~ I l I i ~ ] I I I i I _ _

0 5 10 15 20 25 30 - Alkylput rescine~ m M

12C

11C

100

7O

o 60 '6

~ 4o ©

Fig. 2. Effect of increasing the concentrationsof the N-al- kylputrescines on the activity of rat liver ornithine decarboxy- lase. The incubation mixtures and conditions were the de- scribed in Methods, except for the diamines added. The par- tially purified ammonium sulphate enzymes (0.3 mg) was used. The control had an activity of 4.5_+0.34 nmol 14CO2/mg protein per 40 min. This value was taken as 100% activity. The data shown are the mean values_+ S.D. of five determinations. Putrescine (e . . . . . . e); N-methylputrescine (e e); N- ethylputrescine (O); N-propylputrescine (A); N-butylputres- cine (2x); N, N'-dimethylputrescine (a . . . . . . A); and N,N-di- methylputrescine (A . . . . . . z~).

of the decarboxylase activity at a 25 mM con- centration and showed stronger inhibitions at higher concentrations. Under the same experimen- tal conditions putrescine inhibition leveled off at about 15 mM, when it inhibited 60% of the en- zymatic activity (Fig. 2). The inhibitions exerted by N-propyl- and N,N-dimethylputrescine were of a competitive nature, similar to that of putres- cine.

57

TABLE I

EFFECT OF I-ALKYLPUTRESCINES ON THE ACTIVITY OF RAT LIVER AND E. COLI ORNITHINE DECARBOXY- LASE

The incubation mixtures and conditions were as described in Methods. The control was the standard incubation mixture with no addition. The 1-alkylputrescine was used at a 5 mM final concentration. The data presented are the means+S.D, of six determinations using an enzyme pool prepared from five rats or from 0.5 g of bacteria.

Addition Ornithine decarboxylase activity (nmo114CO2/mg protein per 30 min)

Rat liver E. coli

Inhibition Inhibition (%) (%)

Control 4.5 +- 0.35 - 795_+ 70 - Putrescine 3.3 +- 0.20 28 644_+ 40 19 Methylputrescine 2.7 +_0.20 40 815 + _+ 70 Ethylputrescine 4.0 + 0.15 11 624 + 20 22 n-Propylputrescine 4.5 +_ 0.20 660_+ 30 17 n-Butylputrescine 4.5 _+ 0.20 483 +- 30 39 n-Pentylputrescine 4.15 +0.10 8 515 _+ 30 35 n- Hexylputrosine 3.7 +- 0.10 18 520 _+ 80 35 n-Heptylputrescine 2.7 _+ 0.05 40 350 +_ 20 57

9 O

8 0

7 0

6O

~ 5 0 o

4o

U c5 3© ©

2o!

1C

- ~ ' ""':T " \ " ' r \ \ ~ " - ~ _ ~ Pentylputrescmel r

' ~ , , Hexylputrescine

" "- 1 Putresclne

Heptylputrescine ' ~ f " Methylputresclne ~--

',~

5 10 1 20 25 ~N- Alkylputrescine~ , mM

Effect of C-alkyl putrescines on the activity of ornithine decarboxylase of rat liver and E. coli

T h e ef fec t of an h o m o l o g o u s series of 1-al-

ky lpu t r e sc ines r ang ing f rom ] - m e t h y l p u t r e s c i n e

( 1 , 4 - d i a m i n o p e n t a n e ) to 1 -hep ty lpu t r e sc ine ( l ,4 -

d i a m i n o u n d e c a n e ) was assayed on the ac t iv i ty of

o r n i t h i n e deca rboxy l a se i sola ted f rom rat l iver and

f rom E. coli and their i nh ib i to ry effect was c o m -

p a r e d to that of pu t resc ine . 1 - M e t h y l p u t r e s c i n e

inh ib i t ed the m a m m a l i a n enzyme , whi le it had no

ef fec t on the e n z y m e f rom bac te r i a (Tab le I). As

the cha in length increased , the inh ib i to ry effect on

the l iver e n z y m e dec reased and was nil for 1-

b u t y l p u t r e s c i n e (1 ,4 -d i aminooc tane ) . A n inhib i -

tory effect s ta r ted to a p p e a r aga in f rom 1-penty l -

pu t r e sc ine ( 1 , 4 - d i a m i n o n o n a n e ) onwards , bu t the

assay was d i s c o n t i n u e d af ter using 1-hepty l -

pu t r e sc ine due to the a p p e a r a n c e of de t e rgen t

p rope r t i e s in this c o m p o u n d . T h e la t ter p rope r t i e s

cou ld be r e spons ib le in par t for the inh ib i to ry

Fig. 3. Effect of 1-alkylputrescine concentrations on the activ- ity of the rat liver ornithine decarboxylase. Conditions were as described in Fig. 2. Putrescine (O . . . . . . O); methylputrescine (~ O); ethylputrescine (©); n-butylprescine (z~ zx); n-pentylputrescine (A -,); n-hexylputrescine (A . . . . . . A); and n-heptylputrescine (z~ . . . . . . zx),.

58

effect of several of the a,~-diaminoalkanes used in other studies [12-16], When the above-men- tioned 1-alkylputrescines were assayed using the E. coli decarboxylase, an entirely different inhibi- tory pattern was found (Table I). While 1-methyl- putrescine was not inhibitory, the 1-ethyl deriva- tive had an inhibition similar to that of putrescine and the inhibitory effect increased with chain length. Although the inhibitory effect of the 1- heptyl derivative was noticeably greater than that of its lower homologues, the already mentioned detergent properties of the former could be re- sponsible for the difference. These results lend support to the idea that the active site of the bacterial decarboxylase is different from that of the mammalian enzyme, since it is also known [3] that the 2-difluoromethylornithine is a strong in- hibitor of the liver enzyme but not of the E. coli enzyme.

Increasing concentrations of the 1-alkylputres- cines resulted in a stronger inhibition of the rat liver deca rboxy lase by 1-methyl and 1- heptylputrescine (Fig. 3), while the inhibitory ef- fect of the other diamines did not increase to the same extent with concentration. It should be stressed that l-alkylputrescines are racemates (R, S), and since ornithine decarboxylase is strictly stereospecific [26] it very likely thay only one enantiomer is the active species.

In view of the inhibitory effect of l-methyl- putrescine on the rat liver enzyme and the absence of an inhibitory effect on the bacterial enzyme (Table I), 2-methylputrescine (1,4-diamino-2- methylbutane) was assayed together with the 1,2-, 1,3- and 1,4-dimethylputrescines (Fig. 1). At a 5 mM concentration 2-methylputrescine inhibited 65~, of the activity of the mammalian enzyme and only 25% of the E. coli enzyme. 1,2-Dimethyl- putrescine and 1,3-dimethylputrescine inhibited 27% and 37%, respectively, the activity of the nlammalian decarboxylase, while they inhibited 9% and 43% of the activity of the bacterial de- carboxylase. 1,4-Dimethylputrescine had no in- hibitory effect on the activity of the decarboxylase of either source. These results, taken together with those of Table I, indicate that 2-methylputrescine was the best inhibitor of the rat liver decarboxy- lase among those assayed, while 1,3-dimethyl- putrescine was found to be the best inhibitor of

u i d~

0 30!

0 ©

80 i

~ 2 MethyPputresclne z --- /

' 2L L ~-4 I ~ J B ~ 10i ~ 12 ! 114 I Enh,bitor~ m M

Fig. 4. Effect of increasing concentrations of methyl- and dimethylputrescines on the activity of rat liver ornithine de- carboxylase. Conditions were as in Fig. 2. Putrescine (e . . . . . . e); 1-methylputrescine (e e); 2-methylputrescine (O): 1,2-dimethylputrescine (A); 1,3-dimethylputrescine (zx); and 1,4-dimethylputrescine ( x ).

the bacterial enzyme. Since the synthetic dimethyl- putrescines are a mixture of four isomers (RR, SS, RS and SR) while the synthetic methyl- putrescines are a mixture of two isomers, and if only one of these isomers is the active species, then 2-methylputrescine and 1,3-dimethylputres- cine are good inhibitors of the mammalian en- zyme. Increasing concentrations of the methyldi- amines showed that indeed 2-methylputrescine is among them the best inhibitor of the rat liver enzyme (Fig. 4). The lack of inhibition of 1,4-di- methylputrescine (2,5-diaminohexane) is note- worthy (Fig. 4), and can only be attributed to a steric hindrance in the free rotation of the butane skeleton.

No time-course was observed for the inhibition

TABLE II

INHIBITOR CONSTANTS OF PUTRESCINE AND METHYLPUTRESCINES FOR RAT LIVER ORNITHINE DECARBOXYLASE

The Ki constants were calculated from the data obtained by replotting of the Km,ap p versus inhibitor concentrations.

Inhibitor K i (mM)

Putrescine 3.3 1-Methylputrescine 2.7 2-Methylputrescine 1.0 1,2- Dimethylputrescine 4.8 1,3- Dimet hylputrescine 4.3

of the decarboxylase by the methyl- and dimethyl- putrescine derivatives. Therefore their inhibition was of a competitive nature, as that found for putrescine. 2-Methylputrescine behaved as a typi- cal substrate competitive inhibitor when the re- ciprocal initial velocities (v0) were plotted versus the substrate concentration at different concentra- tions of the inhibitor. Similar curves were ob- tained when 1-methyl-, 1,2-dimethyl- and 1,3-di- methylputrescine were used as inhibitors (data not shown). When the Km,ap p were replotted versus the inhibitor concentrations for the methylputres- cines which acted as inhibitors it was possible to calculate the K~ for the different diamines and compare them with the K~ of putrescine (Table II). A K m = 0.35 mM was found for the rat liver ornithine decarboxylase under the incubation con- ditions used. The data of Table II indicate that a K, = 1 mM was obtained for 2-methylputrescine and since it is very likely that only one of the two enantiomers present in the synthetic material is active, the K~ of 2-methylputrscine would be very similar to the K,,, of the enzyme toward its sub- strate (ornithine). 1,3-Dimethylputrescine was the best inhibitor of the dimethylputrescines assayed (Table II). A K i of 20 mM was found for spermi- dine assayed under the same conditions.

In vivo inhibition of rat liver ornithine decarboxylase activity by methylpurescines

When thioacetamide-treated rats were injected 3 h before death with N-methyl-, 1-methyl- or 2-methylputrescine (50 #mol /100 g body weight) the activity of ornithine decarboxylase was found to be decreased. The decrease in the decarboxylase

59

activity was greater than that exerted by putres- cine under identical conditions and concentra- tions. Decarboxylase activity was assayed on a cytosol fraction (see Methods) which was previ- ously dialyzed to eliminate the polyamines present in this fraction. The absence of polyamines was monitored by dansylation of the extract before and after dialysis. Hence, the in vivo inhibition of the liver ornithine decarboxylase was not due to any residual methylputrescine present in the cyto- sol. 2-Methylputrescine inhibited almost com- pletely the activity of the decarboxylase in vivo at concentrations where putrescine inhibited only 56% of the enzyme's activity (Table IIl). The inhibition found with N-methylputrescine does not reflect the lack of inhibition found with the same derivative on the decarboxylase activity in vitro. The most likely explanation for these inhibitions found in vivo is that the diamines not only inhibit the activity of ornithine decarboxylase but that they also induced the formation of antizymes in vivo [12-16]. They could also interfere with the resynthesis or stability of the ornithine de- carboxylase in the liver, or they could act by a still unknown mechanism, as was suggested when the effect of a number of the commercial a,~-di- amines were assayed in vivo on the induced de- carboxylase activity in mouse epidermis [27].

TABLE Ill

ORNITHINE DECARBOXYLASE ACTIVITY IN RAT LIVER AFTER TREATMENT WITH N-METHYL-, I-METHYL- AND 2-METHYLPUTRESCINE

The animals were treated as described in Methods and ornithine decarboxylase was isolated as described. The 150000x g su- pernatant was dialyzed overnight in the dialysis buffer. The enzymatic activity was assayed as described. The data are the means + S.D. of three experiments with two rats each.

Rats treated Activity Inhibition with (nmo114CO2/mg (%)

protein per 40 min)

Thioacetamide 3.9 _+ 0.20 Thioacetamide plus

putrescine 1.7 _+ 0.20 56 Thioacetamide plus

N-methylputrescine 2.7 +0.15 30 Thioacetamide plus

1-methylputrescine 1.0 + 0.05 75 Thioacetamide plus

2-methylputrescine 0.27 + 0.05 93

60

Discussion

Different synthet ic strategies have been devel- oped to incorpora te into orni th ine (the subs t ra te of orni th ine decarboxylase) , or into putrescine (the enzymat ic react ion product) , latent reactive func-

t ional groups which should result in inhibi tors of this enzyme in efforts d i rected to control poly-

anfine biosynthesis. As we ment ioned above (see In t roduct ion) this research p roduced a number of t ime-dependent (irreversible) inhibi tors of the de- carboxylase and t ime- independent (compet i t ive) inhibi tors of the enzyme. In addi t ion , various di- amines of different chain lengths were assayed and found to inhibi t orni th ine decarboxylase ac- tivity in vivo by the induct ion of an an t ienzyme which in the case of E. coli is very likely a const i tut ive prote in of the r ibosomes [28]. In this repor t we assayed the effect on orn i th ine de- carboxylase , from both a mammal i an and a bacter ial source, of a number of po lyamines which result from a synthet ic app roach where the 1,4-di- a m i n o b u t a n e skeleton of put resc ine remains unchanged and different alkyl subst i tuents are in t roduced into the basis structure. Al though alkyl residues are not very reactive funct ional groups, the results of this s tudy indicate preferred sub- s t i tut ion sites on the put resc ine molecule which results in a greater inhibi tory effect on orn i th ine decarboxylase . The in t roduct ion of alkyl residues on the amino groups of putrescine did not result in compounds which inhibi ted to a great extent the activity of the decarboxylase (Fig. 2). The in t roduct ion of alkyl residues at the C-1 posi t ion of putrescine p roduced d iamines which were com- pet i t ive inhibi tors of the enzyme (Table I and Fig. 3). These results fall in line with the known inhibi- t o r y e f f e c t o f 1 - f l u o r o m e t h y l - a n d 1-ethynylputrescines [7]. The inhibi tory effect of the 1-alkylputrscines was different for the de- carboxylase from rat liver and from E. coli, and this effect was special ly evident for l -me thy l - putrescine (Table I). Subst i tu t ion at the C-2 of putrescine, as in 2-methylput resc ine or 1,3-di- methylput resc ine (see Fig. 1), resulted in bet ter compet i t ive inhibi tors of the enzyme, a useful feature to direct future synthet ic effort.

The fact that N-methylpu t resc ine as well as 1-methyl- and special ly 2-methylput resc ine inhibit

orni th ine decarboxylase act ivi ty in vivo (Table II1) suggests that these d iamines also induce a non-di- a lysable factor which inhibi ts the activi ty of the decarboxylase or affects the s tabi l i ty or resynthe- sis of orni th ine decarboxylase in the liver. There- fore, the C-methylput resc ines have a dual inhibi- tory mechanism for the inhibi t ion of mammal i an orn i th ine decarboxylase , s imilar to but more potent than those exerted by putrescine.

Acknowledgements

This work was made possible by grant G M - 11973 from the N I H (PHS). The help of C O N - ICET (Argent ina) is also acknowledged.

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