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The effects of polyamines on the cyclic amp efflux and metabolism in E. coli b cells
W20-711X/8l~060701-0~102.00/0 Copyright 0 1981 Pergamon Press Ltd
Inr. J. Bxxhum. Vol. 13, pp. 701 to 705. 1981 Punted in Great Br~tam All rights reserved
THE EFFECTS OF POLYAMINES ON THE CYCLIC AMP EFFLUX AND METABOLISM IN E. COLI B CELLS GENNARO ILLIANO, GIULIO
F.
DRAETTA,
ANTONIOLAURENZA,ANNAMARIA SPINA
and GIUSEPPE PAOLISSO
lstituto
di Chimica
Biologica, 1 Facolti Via Costantinopoli
di Medicina e Chirurgia, Universita 16, (I) 80138 Napoli. Italia
(Received 3 Drcember
di Napoli.
1980)
Abstract-l.
The effects of polyamines spermine. spermidine and putrescine have been investigated on the adenylate cyclase and phosphodiesterase activities bound to E. coli B-cell membranes. 2. Putrescine. which is physiologically present in bacteria, affects the’cAMP metabolism in a coordinate manner, depressing the adenylate cyclase and stimulating the phosphodiesterase activities. 1,~ t:ico this is reflected by a decreased CAMP efflux toward the external medium. 3. Spermidine stimulates the phosphodiesterase activities but has no effect on the adenylate cyclase: furthermore no effect was observed iu vice on the CAMP efflux. 4. Spermine stimulates both the enzymic activities but with a prevailing effect on the adenylate cyclase. ln &n the effect is an increase in the CAMP efflux. 5. In cico the intracellular CAMP content is anyway not modified in presence of polyamines, in contrast with (and may be because of) the broad variation of the nucleotide efflux toward the external medium.
INTRODUCTION
third
centrifugation was resuspended in an adequate of saline solution to get a cell number per ml between I5 and 20 x IO’: the bacterial cells were counted in a Biirker apparatus. Each sample was performed in triplicate and contained 2 ml of the cell suspension in a plastic vial; it was preincubated for Smin at 37’C in a shaking Dubnoff incubator and then underwent further incubation in the presence or absence of polyamines. At the times indicated in Table I, the incubation was stopped by rapid filtratioh under vacuum on HA Millipore filters: the filters were immediately washed with 5ml of ice-cold saline solution using a multiple manifold apparatus. The washing step consumed about 10sec. Filtrates and filters have been collected and processed separately for the assay of the CAMP content. Filtrates were added to with 25 pl of 4N HCI. liophylized and redissolved in 0.5 ml of Tris-HCI buffer 50 mM, pH 7.5. The filters were added with 2ml of ice cold saline solution containing 0.2 ml of 50?, trichloroacetic acid (w/v) and sonicated for Zmin at 12A (M.S.E. apparatus). The sonicated materials were centrifuged at 27,ooO 9 for IO min: the supernatant of each sample was added to with 25 ~1 of 4N HCI and extracted 3 times with an equal volume of ether. The aqueous phase was liophylized, redissolved in 0.5 ml of 50 mM Tris-HCl buffer, pH 7.5 and assayed for the CAMP content. volume
Naturally occurring polyamines spermine, spermidine and putrescine can affect many cellular processes also in bacteria and have an important role on the regulation of the cell growth (Tabor & Tabor, 1976; Hirshfield rt al.. 1970; Morris & Jorstad, 1970; Morris & Jorstad, 1973). Many of their biological effects could be explained b> the modulation of the intracellular cyclic AMP (CAMP) content. That’s why we have looked at the effects of the polyamines on the CAMP in E. coli B-ceils as well as on the enzymic activities responsible for its biosynthesis and degradation. Our study refers to short periods (up to 15min) of incubation in the presence of polyamines in a glucose free medium when they are still acting at the membrane level and have not entered the cells (Tabor & Tabor, 1966; Chester et al., 1972; Galanos & Luderitz, 1975). The purpose was to ascribe the observed effects to membrane activities without the complicating interference of intracellular multistep metabolic events.
MATERIALS AND METHODS
CAMP assu~ This was determined on a 50~1 aliquot of the sample and an isotope dilution technique according to Giiman (1970) and Miyamoto er al. (1969). was used, utilizing a specifically CAMP binding protein which we prepared from pig lung (Nakazawa & Sano, 1975). The specificity of the assay was controlled by treating sample quotas with a phosphodiesterase preparation purchased from Boehringer and by previous isolation of CAMP by an ion exchange chromatography on Dowex AG I X8 columns, formate form (Illiano er al.. 1973).
Spermine, spermidine and putrescine were purchased from Sigma Chemical Co. (St Louis. Missouri, U.S.A.); ATP. phosphoenolpyruvate and pyruvate kynasc from Boehringer (West Germany): C3H]cAMP from The Radiochemical Center (Amersham. U.K.). All other reagents were of the highest grade available. In vivo studies E. coli B-cells were cultured on Bacto-Nutrient-Agar plates (Difco Labs. Detroit. U.S.A.), transferred to BactoNutrient-Broth. pH 6.8 and brought into a stationary phase of growth. Before being utilized the cells were washed 3 times with a sterile saline solution and centrifuged at SOOOg for 5 min in the cold. The precipitate of the
En:ymic actiriries related to the CAMP nletabolism
To prepare the enzymic sources. E. coli B-cells were suspended 701
in 50mM
Tris-HCI
buffer.
pH
7.5. containing
GENNARO
702
ILLIAN~ rt al.
1OmM MgCl, and 5 mM mercaptoethanol and sonicated for 2 min at 1.2 A in the cold. The cellular breakage was controlled with a microscope inspection on a Biirker apparatus. After a first centrifugation at 3000 9 for 10 min. the supernatant was centrifuged at 30,COO 9 for 40 min: the precipitate was utilized as enzymic source for the adenylate cyclase activity and for the particulate phosphodiesterase activity. For the adenylate cyclase activity the incubation mixture contained in a final volume of 0.1 ml: 5 mM MgCI,, 1 mM 2.5 mM EDTA, theophylline. 5 mM ATP. 50mM Trls-HCl buffer. pH 7.5. Phosphoenolpyruvate (5 mM) and pyruvate kynase (60 pg/ml) were used as ATP regenerating system. When added, spermine, spermidine and putrescine were diluted in 50mM Tris-HCI buffer, pH 7.5. The reaction was started by adding 15~200 pg of bacterial particulate fraction protein. The samples. prepared in triplicate, were incubated for 1Omin at 3O’C; the reaction was stopped by the addition of 0.1 ml of 0.5 M EDTA. pH 7.5, immediately followed by a 3 min boiling; samples were added with 0.8 ml of ice cold 50 mM Tris-HCl buffer. pH 7.5 and centrifuged in cold at 10,000 9 for 10 min. For each sample 50 ~1 of the supernatant were used to determine the CAMP content. Kinetic experiments were carried out in presence of [z--“P]ATP (1.5&i) and various amounts of unlabeled ATP. as indicated in the legends of figures. C3H]cAMP (24 Ci/mmol) equivalent to 30,000 cpm was added for the determination of recovery. The formed [32P]cAMP was isolated according to Salomon rt al. (1974). The incubation mixture for the phosphodiesterase activity contained in a final volume of 0.1 ml: 50mM Tris-HCl buffer. pH 7.5, 2 mM EDTA, 9 mM MgCl,. CAMP as indicated (10m6 or 10-j M), C3H]cAMP (24 Ci/ mmol) equivalent to 50,OOOcpm. for the determination of recovery. Spermine. spermidine and putrescine, when added. were diluted in 50 mM Tris-HCI, pH 7.5. The reaction was started by the addition of the particulate enzymic source (1 O& 150 pg of protein). The incubation was carried out at 37-C for 1Omin and stopped by the addition of 0.1 ml of 0.5 M EDTA, pH 7.5, immediately followed by boiling for 3min. After centrifugation for 10min at 10,000 IJ. the supernatant of each sample was added to with 0.8 ml of 50mM Tris+HCl buffer. pH 7.5 and passed through a alumina column (1 g of neutral alumina in a 0.5 x 2cm column) previously equilibrated with the same buffer; the residual C3H]cAMP was eluted with 3 ml of the same buffer (Ramachandran, 1971; White & Zenser. 1971) and counted after the addition of 9ml of Packard Picofluor scintillation medium.
Table
1. Intra-
and
Time (min)
0 5 10 15
extracellular
distributions
RESULTS
Extra 359 * 368 + 381 + 321 *
30 28 31 40
Intra 6+1 6+1 5+2 7+1
DISCUSSIOIV
Table 1 shows that there is an active CAMP efflux toward the extracellular medium in the first minutes following the explant. while the intracellular CAMP is quite constant, indicating that during this period the CAMP biosynthesis compensates either the breakdown or the outside loss. The presence of polyamines didn’t modify the timecourse of the intracellular CAMP content, but deeply modified the one of the extracellular CAMP, in a way characteristic for each polyamine. In fact the presence of lo- ’ M spermine in the medium brought about a much higher CAMP content; on the contrary. the addition of IO- ’ M putrescine brought about a decrease of the CAMP content. The addition of lo-” M spermidine didn’t give different results to the controls. These observations refer to short periods of incubation, on a glucose-free medium when polyamines are supposed to act at a membrane level, since their intracellular transport has been demonstrated to be carrier mediated and energy requiring (Tabor & Tabor. 1966; Chester er al., 1972: Galanos & Luderitz, 1975). The effects of polyamines on the membrane-bound envymic activities related to the CAMP biosynthesis and degradation An appreciable adenylate cyclase activity is present in the particulate cell fraction obtained from E. co/i B-cells and corresponds approx to 87 pmol of CAMP formed/mg protein/min _ ’ in presence of 5 mM ATP (value 100 of Fig. 1). As already shown for eucaryotic cell membranes (Illiano et al., 1979) the polyamine effects depend on the type as well as on the concentration of the added polyamine; in fact putrescine inhibits the adenylate
of CAMP in E. co/i B-cells transplantation
Extra 389 + 399 + 586 + 318 *
AND
The effects of polyamines spermine and putrescine on the time course of the intracellular and extracellular CAMP content in a saline suspension of‘ E. co/i B-cells
+ Spermine 10-5M CAMP content,
None Intra 9*2 6? 1 6il 8i2
A polyamine effect on the CAMP elution profile on the alumina columns was ruled out by controlling the [‘HIcAMP chromatographic recovery from samples incubated in presence of boiled enzymic source with and without added polyamines.
40 35 38 31
during
the
+ Spermidine 10-5M pmol lntra 5+1 6kl 6+1 8*1
Extra 360 & 355 + 387 + 298 +
early
phases
of culture
+ Putrescine 10-5M
31 30 32 30
Intra 7+1 9+7 9+2 7*1
Extra 350 f 180 f 150 + 280 f.
30 20 21 30
E. co/i B-cells, in a stationary phase of growth, were transferred from a peptonized growth medium into 2 ml of saline, solution, after being washed 3 times with the same solution. Each sample contained approx 20 x lo6 cells in a plastic vial: after a 5 min period of preincubation, at 37”C, in a shaking Dubnoff incubator. cell suspensions were further incubated in the presence or absence of polyamines. The incubation was stopped by rapid filtration under vacuum HA Millipore filters; the filters were immediately washed with 5 ml of ice-cold saline solution using a multiple manifold apparatus. The washing step consumed about 10sec. For other experimental conditions. see the text. The data indicate the results (average + SEM) of 2 separate experiments, performed in triplicate.
Polyamines affect E.
co/i
CAMP efflux
%
703
-b Log
--it
-6
Log
-5
polyamine
-4
odded, M
Fig. 1. Influence of polyamines, at different concentrations, on the E. co/i B-cells adenylate cyclase activity. Results are indicated as percentage of the controls. 100’~ corresponds to 87 pimol of CAMP formed/mg protein/min-‘. (+a), + spermine; (V---V), + spermidine; (WA), + putrescine. Each point is the mean of 3-5 experiments, performed in triplicate. For the experimental conditions see the text.
cyclase activity at a very low concentration but it is less effective or completely ineffective at higher concentrations; spermine, on the contrary, stimulates the
-
XI0
-;
i -4
spermine ,
-3
-2
M
Fig. 2. Effect of spermine concentration in presence of two substrate concentrations, saturating or not, on the activation of the E. co/i B-cell adenylate cyclase activity. Each point is the mean of 3-5 experiments, performed in tripicate. For the experimental conditions see the text.
cyclase activity with a linear dose-effect relationship over a large range of concentrations, reaching the plateau between 10e4 and IO- ’ M in presence of both low and saturating concentrations of the substrate (Fig. 2). Spermidine, at the tested concentrations (Fig. I), didn’t affect the enzymic activity. As to the activating effect of spermine, the kinetics carried out in presence of two different substrate concentrations and several spermine concentrations
-3
ATP ( M)
Fig. 3. E. coli B-cell anenylate cyclase activity: double reciprocal plot of the reaction in the absence and in the presence of different spermine concentrations. (*o), controls: (M )_+ spermine. For the experimental conditions see the text.
GENNARO
704
ILLIANO et cd.
I-
b
:&W’l
(cAh4P)IO%
lO+M
Put SP
I
T SP
-: Fig. 4. Influence of polyamines on the particulate phosphodiesterases activities in E. coii B-cells. The experiments have been carried out in presence of two CAMP concentrations. For each CAMP concentration the results are expressed as percentage of the basal (without addition) activity: (a) lOweM CAMP: the basal activity corresponds to 14 pmol of CAMP hydrolyzed/mg protein/min-’ (value 100 in the figure): (b) 10e3 M CAMP: the basal activity corresponds to 318 pmol of CAMP hydrolyzed/mg protein/mine’ (value 100 in the figure). No. basal activity; Sp, + 10e5M spermine: Spd. + lo-” M spermidine; put, + toe5 M putrescine. Bars indicate the SEM of 3 separate experiments, performed in triplicate.
(Fig. 2) show the activating effect to be independent of the substrate concentration, but strictly dependent of the spermine one. The kinetics related in Fig. 3 show that the apparent K, results in a decrease only in presence of spermine higher concentrations. while it is immodified at spermine lower ones. To explain such effects we could admit that a spermine-ATP complex or spermine by itself, could act as a positive effector on the enzyme. Moreover a polyamine interaction with opposite charged membrane components is likely to modify either the membrane tlui,dity or the exposure and the accessibility of the adenylate cyclase system or its affinity for the Mg-ATP complex or the spermine-ATP one (Rodbell, 1980). Polyamines have been supposed to be able to compete with the Mg” ions in forming complexes with ATP, owing to the high concentrations they can reach inside the cell and the high association constant for ATP at the cellular pH (Nakai & Glinsmann, 1977); for putrescine, the formation of the polyamine--ATP complex instead of Mg-ATP, which is the true substrate for the adenylate cyclase activity. could be the molecular basis of the inhibitory effect. Without excluding it. our data show the operativeness also of a polyamine direct effect on the adenylate cyclase system: in fact, the putrescine inhibitory effect is obtained in absence of intracellular polyamines. in presence of a putrescine concentration which is very low compared with the ATP one and in presence of adequate Mg2+ concentrations. The inhibitory effect on the adenylate cyclase activity by putrescine could have been only apparent but truly due to the stimulating effect exerted by the polyamine on the membrane-bound phosphodiester-
ase activities (see forward, Fig. 4). Moreover we were aware that bacterial phosphodiesterase have been described as insensitive to classicai xanthinic inhibitors (Rickenberg, 1974). Thought the decreasing of the inhibitory effect in presence of higher putrescine concentrations makes unlikely a substantial contribution to be brought by such a mechanism. experiments have been performed in presence of labelled [K-~~PJATP aud a large pool (IOOmM) of unlabelled CAMP in order to minimize the effects of phosphodiesterase activities. Also in these conditions the presence of lo-’ M putrescine clearly inhibited the adenylate cycIas- activity. Basically the same results were obtained in the absence of the ATP regenerating system over a 5 min period of incubation. The polyamines, up to lo-* M didn’t affect the ATP stability as controlled by thin-layer chromatography on silica gel plates (Merck F2.54) using isopropyl alcohol-water-ammonium hydroxide (7 : 2: 1) as a solvent mixture (Monard et al., 1969). As to the effects on the particulate phosphodiesterase activities (Monard ef al., 1969). the results are shown in Fig. 4; they indicate that at the lowest (IO-” M) CAMP concentration the hydrolytic activity corresponds to 14 pimol of S-AMP formed/mg protein/min - ’ and it is greatly stimulated by polyamines. The highest (IO- 3 M) CAMP concentration greatly increases the enzymic activity by itself (S.A. 318 pimol/mg protein/mine’1 and can be slightly stimulated only by putrescine. At present it is not possible to say whether in presence of higher substrate concentrations other populations of phosphodiesterases, with a lower affinity for CAMP become operative, or the CAMP acts, at higher concentrations.
Polyamines affect E. co/i CAMP efflux as a positive effector minimizing and masking the positive effect due to polyamines. The products of CAMP hydrolysis, in the absence and in the presence of polyamines, were analyzed by thin-layer chromatography on silica gel plates (Monard et al., 1969). The results (data not shown) indicate that polyamines do not affect the CAMP stability and that the enzymic hydrolysis of CAMP is followed by the further degradation giving rise to the formation of adenosine and inosine. In conclusion these results indicate that putrescine. a polyamine naturally occurring in bacteria, acts sinergistically depressing the adenylate cyclase acti\ ity and stimulating the phosphodiesterase activities; for spermine. which is virtually absent in bacteria (Tabor & Tabor, 1976), the stimulating effect on the adenylate cyclase activity prevails on the same effect on the phosphodiesterase activities. As far as spermidine is concerned, we cannot explain why the remarkable stimulating effect on the phosphodiesterase activities. shown in citro doesn’t affect at least the extracellular CAMP content when spermidine is added to living cells. The heavy perturbations apported by polyamines on the CAMP metabolism are any way without effect irk rive on the intracellular CAMP content. in the experimental conditions we used (Table I): our study demonstrates that in bacterial cells CAMP efflux (Monard et al.. 1969; Buettner et al.. 1973; Konijn er al.. 1969) is finely regulated and can keep at a steadystate level the intracellular nucleotide concentrations. REFERENCES BLFTTNERM. J.. SPITZE. & RICKENBERG H. V. (1973) CycIICadenosine 3’S’-monophosphate in Eschericllia coli. J. &ICI. 114. 1068-1073. CHESTER 1. R., GRAY G. W.. WILKINSONS. G. (1972) Further studies on the chemical composition of lipopolysaccharide of Pseudomonas aeruginosa. Eiochem. J. 126. .795-407. GALANOSC. & LUDERITZ0. (1975) Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur. J. Biochem. 54, 603-610. GIL.MANA. G. (1970) A protein binding assay for adenosine 3’S’-cyclic monophosphate. Proc. natn. Acad. Sci. U.S.A. 67. 305-3
17.
HIKSHFIELD I. N., ROSENFELD H., LEIFERZ. & MAASW. K. (1970) Isolation and characterisation of a mutant of Escherichia co/i blocked in the synthesis of putrescine. J. Bact.
101. 725-730.
K~VIJN T. M.. VAN DE MEEMEJ. G. C..
CHANG Y. Y..
705
BARKLEYD. S. & BONERJ. T. (1969) Identification of adenosine 3’,5’-monophosphate as the bacterial attractant for mixoamoebae of Dictyostelium Discoideum. J. Bact. 99. 5 I O-5 12. ILLIANOG.. TELLG. P. E.. SEGELM. E. & CUATRECASAS P. (1973) Guanosine 3’.5’-monophosphate and the action of insulin and acetilcholine. Proc. natn. Acad. Sci. U.S.A. 70. 2443-2447. ILLIANOG.. SPINA A.,
LAURENZAA.. DRAETTAG. F., GALDERISIR., CHIOSIE. (1979) The influence of polyamines on the cyclic AMP metabolism in rat liver slices. Bull. molec. Biol. Med. 4. 155-165. MIYAMOTO E., Kuo J. F. & GREENGARD (1969) Cyclic nucleotide-dependent protein kynases. III. Purification and properties of adenosine 3’.5’-monophosphate-dependent protein kynase from bovine brain. J. ho/. Chem. 244, 6395-6401. MONARDD.. JANACE~:J. & RICKENBERG H. V. (1969) The enzymic degradation of 3’.5’-cyclic AMP in strains sensitive and resistants to cataboli!e repression. Biochem. hiophys. Rex Commun.
35, 584591.
MORRISD. R. & JORSTADC. M. (1970) Isolation of conditionally putrescine deficient mutants of Escherichia coli. J. Bact.
101. 731-737.
MORRISD. R. & JORSTADC. M. (I 973) Growth and macromolecular composition of a mutant of E. co/i during polyamine limitation. J. Bact. 113, 27lL277. NAKAI C. & GLINSMANNW. (1977) Interaction between polyamines and nucleotides. Biochemistry 16, 56365640. NAKAZAWAK. B S.+NOM. (1975) Partial purification and properties of guanosine 3’.5’-monophosphate-dependent protein kynase from pig lung. J. hiol. Ckem. 250, 7415-7419.
RAMACHANDRAN J. (1971) A new simple method for separation of adenosine 3’.5’-monophosphate from other nucleotides and its use in the assay of adenyl cyclase. AnaIyt. Biochem.
43, 227-239.
RICI(ENBERG H. V. (1974) Cyclic AMP in prokaryotes. Rev. Microhiol.
A.
28, 353-369.
RODBELLM. (1980) The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature
284. 17-22.
SALOMON Y., LONDOSC.. RODBELLM. (1974) A highly sensitive adenylate cyclase assay. A,lalyt. Biochem. 58, 541-548.
TABORC. W. & TABOR H. (1966) Transport systems for l,4-diaminobutane, spermidine and spermine in Escherichin co/i. .I. Biol. Chem. 241. 37143723. TABOR C. W. & TABOR H. (1976) l.4-diaminobutane (putrescine), spermidine and spermine. A. Rec. Biochem. 45, 285-306. WHITE A. A. & ZENSERT. V. (1971) Separation of cyclic 3’,5’-nucleoside monophosphate from other nucleotides on aluminium oxide columns. Analyt. Biochem. 41, 372-396.
Inr. J. Bxxhum. Vol. 13, pp. 701 to 705. 1981 Punted in Great Br~tam All rights reserved
THE EFFECTS OF POLYAMINES ON THE CYCLIC AMP EFFLUX AND METABOLISM IN E. COLI B CELLS GENNARO ILLIANO, GIULIO
F.
DRAETTA,
ANTONIOLAURENZA,ANNAMARIA SPINA
and GIUSEPPE PAOLISSO
lstituto
di Chimica
Biologica, 1 Facolti Via Costantinopoli
di Medicina e Chirurgia, Universita 16, (I) 80138 Napoli. Italia
(Received 3 Drcember
di Napoli.
1980)
Abstract-l.
The effects of polyamines spermine. spermidine and putrescine have been investigated on the adenylate cyclase and phosphodiesterase activities bound to E. coli B-cell membranes. 2. Putrescine. which is physiologically present in bacteria, affects the’cAMP metabolism in a coordinate manner, depressing the adenylate cyclase and stimulating the phosphodiesterase activities. 1,~ t:ico this is reflected by a decreased CAMP efflux toward the external medium. 3. Spermidine stimulates the phosphodiesterase activities but has no effect on the adenylate cyclase: furthermore no effect was observed iu vice on the CAMP efflux. 4. Spermine stimulates both the enzymic activities but with a prevailing effect on the adenylate cyclase. ln &n the effect is an increase in the CAMP efflux. 5. In cico the intracellular CAMP content is anyway not modified in presence of polyamines, in contrast with (and may be because of) the broad variation of the nucleotide efflux toward the external medium.
INTRODUCTION
third
centrifugation was resuspended in an adequate of saline solution to get a cell number per ml between I5 and 20 x IO’: the bacterial cells were counted in a Biirker apparatus. Each sample was performed in triplicate and contained 2 ml of the cell suspension in a plastic vial; it was preincubated for Smin at 37’C in a shaking Dubnoff incubator and then underwent further incubation in the presence or absence of polyamines. At the times indicated in Table I, the incubation was stopped by rapid filtratioh under vacuum on HA Millipore filters: the filters were immediately washed with 5ml of ice-cold saline solution using a multiple manifold apparatus. The washing step consumed about 10sec. Filtrates and filters have been collected and processed separately for the assay of the CAMP content. Filtrates were added to with 25 pl of 4N HCI. liophylized and redissolved in 0.5 ml of Tris-HCI buffer 50 mM, pH 7.5. The filters were added with 2ml of ice cold saline solution containing 0.2 ml of 50?, trichloroacetic acid (w/v) and sonicated for Zmin at 12A (M.S.E. apparatus). The sonicated materials were centrifuged at 27,ooO 9 for IO min: the supernatant of each sample was added to with 25 ~1 of 4N HCI and extracted 3 times with an equal volume of ether. The aqueous phase was liophylized, redissolved in 0.5 ml of 50 mM Tris-HCl buffer, pH 7.5 and assayed for the CAMP content. volume
Naturally occurring polyamines spermine, spermidine and putrescine can affect many cellular processes also in bacteria and have an important role on the regulation of the cell growth (Tabor & Tabor, 1976; Hirshfield rt al.. 1970; Morris & Jorstad, 1970; Morris & Jorstad, 1973). Many of their biological effects could be explained b> the modulation of the intracellular cyclic AMP (CAMP) content. That’s why we have looked at the effects of the polyamines on the CAMP in E. coli B-ceils as well as on the enzymic activities responsible for its biosynthesis and degradation. Our study refers to short periods (up to 15min) of incubation in the presence of polyamines in a glucose free medium when they are still acting at the membrane level and have not entered the cells (Tabor & Tabor, 1966; Chester et al., 1972; Galanos & Luderitz, 1975). The purpose was to ascribe the observed effects to membrane activities without the complicating interference of intracellular multistep metabolic events.
MATERIALS AND METHODS
CAMP assu~ This was determined on a 50~1 aliquot of the sample and an isotope dilution technique according to Giiman (1970) and Miyamoto er al. (1969). was used, utilizing a specifically CAMP binding protein which we prepared from pig lung (Nakazawa & Sano, 1975). The specificity of the assay was controlled by treating sample quotas with a phosphodiesterase preparation purchased from Boehringer and by previous isolation of CAMP by an ion exchange chromatography on Dowex AG I X8 columns, formate form (Illiano er al.. 1973).
Spermine, spermidine and putrescine were purchased from Sigma Chemical Co. (St Louis. Missouri, U.S.A.); ATP. phosphoenolpyruvate and pyruvate kynasc from Boehringer (West Germany): C3H]cAMP from The Radiochemical Center (Amersham. U.K.). All other reagents were of the highest grade available. In vivo studies E. coli B-cells were cultured on Bacto-Nutrient-Agar plates (Difco Labs. Detroit. U.S.A.), transferred to BactoNutrient-Broth. pH 6.8 and brought into a stationary phase of growth. Before being utilized the cells were washed 3 times with a sterile saline solution and centrifuged at SOOOg for 5 min in the cold. The precipitate of the
En:ymic actiriries related to the CAMP nletabolism
To prepare the enzymic sources. E. coli B-cells were suspended 701
in 50mM
Tris-HCI
buffer.
pH
7.5. containing
GENNARO
702
ILLIAN~ rt al.
1OmM MgCl, and 5 mM mercaptoethanol and sonicated for 2 min at 1.2 A in the cold. The cellular breakage was controlled with a microscope inspection on a Biirker apparatus. After a first centrifugation at 3000 9 for 10 min. the supernatant was centrifuged at 30,COO 9 for 40 min: the precipitate was utilized as enzymic source for the adenylate cyclase activity and for the particulate phosphodiesterase activity. For the adenylate cyclase activity the incubation mixture contained in a final volume of 0.1 ml: 5 mM MgCI,, 1 mM 2.5 mM EDTA, theophylline. 5 mM ATP. 50mM Trls-HCl buffer. pH 7.5. Phosphoenolpyruvate (5 mM) and pyruvate kynase (60 pg/ml) were used as ATP regenerating system. When added, spermine, spermidine and putrescine were diluted in 50mM Tris-HCI buffer, pH 7.5. The reaction was started by adding 15~200 pg of bacterial particulate fraction protein. The samples. prepared in triplicate, were incubated for 1Omin at 3O’C; the reaction was stopped by the addition of 0.1 ml of 0.5 M EDTA. pH 7.5, immediately followed by a 3 min boiling; samples were added with 0.8 ml of ice cold 50 mM Tris-HCl buffer. pH 7.5 and centrifuged in cold at 10,000 9 for 10 min. For each sample 50 ~1 of the supernatant were used to determine the CAMP content. Kinetic experiments were carried out in presence of [z--“P]ATP (1.5&i) and various amounts of unlabeled ATP. as indicated in the legends of figures. C3H]cAMP (24 Ci/mmol) equivalent to 30,000 cpm was added for the determination of recovery. The formed [32P]cAMP was isolated according to Salomon rt al. (1974). The incubation mixture for the phosphodiesterase activity contained in a final volume of 0.1 ml: 50mM Tris-HCl buffer. pH 7.5, 2 mM EDTA, 9 mM MgCl,. CAMP as indicated (10m6 or 10-j M), C3H]cAMP (24 Ci/ mmol) equivalent to 50,OOOcpm. for the determination of recovery. Spermine. spermidine and putrescine, when added. were diluted in 50 mM Tris-HCI, pH 7.5. The reaction was started by the addition of the particulate enzymic source (1 O& 150 pg of protein). The incubation was carried out at 37-C for 1Omin and stopped by the addition of 0.1 ml of 0.5 M EDTA, pH 7.5, immediately followed by boiling for 3min. After centrifugation for 10min at 10,000 IJ. the supernatant of each sample was added to with 0.8 ml of 50mM Tris+HCl buffer. pH 7.5 and passed through a alumina column (1 g of neutral alumina in a 0.5 x 2cm column) previously equilibrated with the same buffer; the residual C3H]cAMP was eluted with 3 ml of the same buffer (Ramachandran, 1971; White & Zenser. 1971) and counted after the addition of 9ml of Packard Picofluor scintillation medium.
Table
1. Intra-
and
Time (min)
0 5 10 15
extracellular
distributions
RESULTS
Extra 359 * 368 + 381 + 321 *
30 28 31 40
Intra 6+1 6+1 5+2 7+1
DISCUSSIOIV
Table 1 shows that there is an active CAMP efflux toward the extracellular medium in the first minutes following the explant. while the intracellular CAMP is quite constant, indicating that during this period the CAMP biosynthesis compensates either the breakdown or the outside loss. The presence of polyamines didn’t modify the timecourse of the intracellular CAMP content, but deeply modified the one of the extracellular CAMP, in a way characteristic for each polyamine. In fact the presence of lo- ’ M spermine in the medium brought about a much higher CAMP content; on the contrary. the addition of IO- ’ M putrescine brought about a decrease of the CAMP content. The addition of lo-” M spermidine didn’t give different results to the controls. These observations refer to short periods of incubation, on a glucose-free medium when polyamines are supposed to act at a membrane level, since their intracellular transport has been demonstrated to be carrier mediated and energy requiring (Tabor & Tabor. 1966; Chester er al., 1972: Galanos & Luderitz, 1975). The effects of polyamines on the membrane-bound envymic activities related to the CAMP biosynthesis and degradation An appreciable adenylate cyclase activity is present in the particulate cell fraction obtained from E. co/i B-cells and corresponds approx to 87 pmol of CAMP formed/mg protein/min _ ’ in presence of 5 mM ATP (value 100 of Fig. 1). As already shown for eucaryotic cell membranes (Illiano et al., 1979) the polyamine effects depend on the type as well as on the concentration of the added polyamine; in fact putrescine inhibits the adenylate
of CAMP in E. co/i B-cells transplantation
Extra 389 + 399 + 586 + 318 *
AND
The effects of polyamines spermine and putrescine on the time course of the intracellular and extracellular CAMP content in a saline suspension of‘ E. co/i B-cells
+ Spermine 10-5M CAMP content,
None Intra 9*2 6? 1 6il 8i2
A polyamine effect on the CAMP elution profile on the alumina columns was ruled out by controlling the [‘HIcAMP chromatographic recovery from samples incubated in presence of boiled enzymic source with and without added polyamines.
40 35 38 31
during
the
+ Spermidine 10-5M pmol lntra 5+1 6kl 6+1 8*1
Extra 360 & 355 + 387 + 298 +
early
phases
of culture
+ Putrescine 10-5M
31 30 32 30
Intra 7+1 9+7 9+2 7*1
Extra 350 f 180 f 150 + 280 f.
30 20 21 30
E. co/i B-cells, in a stationary phase of growth, were transferred from a peptonized growth medium into 2 ml of saline, solution, after being washed 3 times with the same solution. Each sample contained approx 20 x lo6 cells in a plastic vial: after a 5 min period of preincubation, at 37”C, in a shaking Dubnoff incubator. cell suspensions were further incubated in the presence or absence of polyamines. The incubation was stopped by rapid filtration under vacuum HA Millipore filters; the filters were immediately washed with 5 ml of ice-cold saline solution using a multiple manifold apparatus. The washing step consumed about 10sec. For other experimental conditions. see the text. The data indicate the results (average + SEM) of 2 separate experiments, performed in triplicate.
Polyamines affect E.
co/i
CAMP efflux
%
703
-b Log
--it
-6
Log
-5
polyamine
-4
odded, M
Fig. 1. Influence of polyamines, at different concentrations, on the E. co/i B-cells adenylate cyclase activity. Results are indicated as percentage of the controls. 100’~ corresponds to 87 pimol of CAMP formed/mg protein/min-‘. (+a), + spermine; (V---V), + spermidine; (WA), + putrescine. Each point is the mean of 3-5 experiments, performed in triplicate. For the experimental conditions see the text.
cyclase activity at a very low concentration but it is less effective or completely ineffective at higher concentrations; spermine, on the contrary, stimulates the
-
XI0
-;
i -4
spermine ,
-3
-2
M
Fig. 2. Effect of spermine concentration in presence of two substrate concentrations, saturating or not, on the activation of the E. co/i B-cell adenylate cyclase activity. Each point is the mean of 3-5 experiments, performed in tripicate. For the experimental conditions see the text.
cyclase activity with a linear dose-effect relationship over a large range of concentrations, reaching the plateau between 10e4 and IO- ’ M in presence of both low and saturating concentrations of the substrate (Fig. 2). Spermidine, at the tested concentrations (Fig. I), didn’t affect the enzymic activity. As to the activating effect of spermine, the kinetics carried out in presence of two different substrate concentrations and several spermine concentrations
-3
ATP ( M)
Fig. 3. E. coli B-cell anenylate cyclase activity: double reciprocal plot of the reaction in the absence and in the presence of different spermine concentrations. (*o), controls: (M )_+ spermine. For the experimental conditions see the text.
GENNARO
704
ILLIANO et cd.
I-
b
:&W’l
(cAh4P)IO%
lO+M
Put SP
I
T SP
-: Fig. 4. Influence of polyamines on the particulate phosphodiesterases activities in E. coii B-cells. The experiments have been carried out in presence of two CAMP concentrations. For each CAMP concentration the results are expressed as percentage of the basal (without addition) activity: (a) lOweM CAMP: the basal activity corresponds to 14 pmol of CAMP hydrolyzed/mg protein/min-’ (value 100 in the figure): (b) 10e3 M CAMP: the basal activity corresponds to 318 pmol of CAMP hydrolyzed/mg protein/mine’ (value 100 in the figure). No. basal activity; Sp, + 10e5M spermine: Spd. + lo-” M spermidine; put, + toe5 M putrescine. Bars indicate the SEM of 3 separate experiments, performed in triplicate.
(Fig. 2) show the activating effect to be independent of the substrate concentration, but strictly dependent of the spermine one. The kinetics related in Fig. 3 show that the apparent K, results in a decrease only in presence of spermine higher concentrations. while it is immodified at spermine lower ones. To explain such effects we could admit that a spermine-ATP complex or spermine by itself, could act as a positive effector on the enzyme. Moreover a polyamine interaction with opposite charged membrane components is likely to modify either the membrane tlui,dity or the exposure and the accessibility of the adenylate cyclase system or its affinity for the Mg-ATP complex or the spermine-ATP one (Rodbell, 1980). Polyamines have been supposed to be able to compete with the Mg” ions in forming complexes with ATP, owing to the high concentrations they can reach inside the cell and the high association constant for ATP at the cellular pH (Nakai & Glinsmann, 1977); for putrescine, the formation of the polyamine--ATP complex instead of Mg-ATP, which is the true substrate for the adenylate cyclase activity. could be the molecular basis of the inhibitory effect. Without excluding it. our data show the operativeness also of a polyamine direct effect on the adenylate cyclase system: in fact, the putrescine inhibitory effect is obtained in absence of intracellular polyamines. in presence of a putrescine concentration which is very low compared with the ATP one and in presence of adequate Mg2+ concentrations. The inhibitory effect on the adenylate cyclase activity by putrescine could have been only apparent but truly due to the stimulating effect exerted by the polyamine on the membrane-bound phosphodiester-
ase activities (see forward, Fig. 4). Moreover we were aware that bacterial phosphodiesterase have been described as insensitive to classicai xanthinic inhibitors (Rickenberg, 1974). Thought the decreasing of the inhibitory effect in presence of higher putrescine concentrations makes unlikely a substantial contribution to be brought by such a mechanism. experiments have been performed in presence of labelled [K-~~PJATP aud a large pool (IOOmM) of unlabelled CAMP in order to minimize the effects of phosphodiesterase activities. Also in these conditions the presence of lo-’ M putrescine clearly inhibited the adenylate cycIas- activity. Basically the same results were obtained in the absence of the ATP regenerating system over a 5 min period of incubation. The polyamines, up to lo-* M didn’t affect the ATP stability as controlled by thin-layer chromatography on silica gel plates (Merck F2.54) using isopropyl alcohol-water-ammonium hydroxide (7 : 2: 1) as a solvent mixture (Monard et al., 1969). As to the effects on the particulate phosphodiesterase activities (Monard ef al., 1969). the results are shown in Fig. 4; they indicate that at the lowest (IO-” M) CAMP concentration the hydrolytic activity corresponds to 14 pimol of S-AMP formed/mg protein/min - ’ and it is greatly stimulated by polyamines. The highest (IO- 3 M) CAMP concentration greatly increases the enzymic activity by itself (S.A. 318 pimol/mg protein/mine’1 and can be slightly stimulated only by putrescine. At present it is not possible to say whether in presence of higher substrate concentrations other populations of phosphodiesterases, with a lower affinity for CAMP become operative, or the CAMP acts, at higher concentrations.
Polyamines affect E. co/i CAMP efflux as a positive effector minimizing and masking the positive effect due to polyamines. The products of CAMP hydrolysis, in the absence and in the presence of polyamines, were analyzed by thin-layer chromatography on silica gel plates (Monard et al., 1969). The results (data not shown) indicate that polyamines do not affect the CAMP stability and that the enzymic hydrolysis of CAMP is followed by the further degradation giving rise to the formation of adenosine and inosine. In conclusion these results indicate that putrescine. a polyamine naturally occurring in bacteria, acts sinergistically depressing the adenylate cyclase acti\ ity and stimulating the phosphodiesterase activities; for spermine. which is virtually absent in bacteria (Tabor & Tabor, 1976), the stimulating effect on the adenylate cyclase activity prevails on the same effect on the phosphodiesterase activities. As far as spermidine is concerned, we cannot explain why the remarkable stimulating effect on the phosphodiesterase activities. shown in citro doesn’t affect at least the extracellular CAMP content when spermidine is added to living cells. The heavy perturbations apported by polyamines on the CAMP metabolism are any way without effect irk rive on the intracellular CAMP content. in the experimental conditions we used (Table I): our study demonstrates that in bacterial cells CAMP efflux (Monard et al.. 1969; Buettner et al.. 1973; Konijn er al.. 1969) is finely regulated and can keep at a steadystate level the intracellular nucleotide concentrations. REFERENCES BLFTTNERM. J.. SPITZE. & RICKENBERG H. V. (1973) CycIICadenosine 3’S’-monophosphate in Eschericllia coli. J. &ICI. 114. 1068-1073. CHESTER 1. R., GRAY G. W.. WILKINSONS. G. (1972) Further studies on the chemical composition of lipopolysaccharide of Pseudomonas aeruginosa. Eiochem. J. 126. .795-407. GALANOSC. & LUDERITZ0. (1975) Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur. J. Biochem. 54, 603-610. GIL.MANA. G. (1970) A protein binding assay for adenosine 3’S’-cyclic monophosphate. Proc. natn. Acad. Sci. U.S.A. 67. 305-3
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RAMACHANDRAN J. (1971) A new simple method for separation of adenosine 3’.5’-monophosphate from other nucleotides and its use in the assay of adenyl cyclase. AnaIyt. Biochem.
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TABORC. W. & TABOR H. (1966) Transport systems for l,4-diaminobutane, spermidine and spermine in Escherichin co/i. .I. Biol. Chem. 241. 37143723. TABOR C. W. & TABOR H. (1976) l.4-diaminobutane (putrescine), spermidine and spermine. A. Rec. Biochem. 45, 285-306. WHITE A. A. & ZENSERT. V. (1971) Separation of cyclic 3’,5’-nucleoside monophosphate from other nucleotides on aluminium oxide columns. Analyt. Biochem. 41, 372-396.