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Induction of an ornithine decarboxylase-antizyme in chicken liver
,,l, J B,,,‘h‘W.. Vol I I. pp. 37 10 42 0 Pcrgamon Presc Ltd 1980. Prmted m Great Bmain
INDUCTION OF AN ORNITHINE DECARBOXYLASE-ANTIZYME IN CHICKEN M. A. Cattedra
di Chimica
GRILLO,
S. BEDINO
LIVER
and G. TESTORE
e Istituto di Chimica biologica della Facolta dell’Universit8 di Torino, Torino, Italy
di Medicina
e Chirurgia
(Rrceived 18 May 1979) Abstract---l. The insulin-induced increase in ornithine decarboxylase and in S-adenosylmethionine decarboxylase in chicken liver is considerably inhibited by diaminopropane. This effect does not last long: after 5 hr, ornithine decarboxylase has returned to normal, though S-adenosylmethionine decarboxylase is more active. 2. There is a good correlation between diaminopropane and ornithine decarboxylase activity for I hr. after which ornithine decarboxylase remains low, even when diaminopropane disappears from the liver. 3. Liver diaminobutane concentration is fairly constant throughout the treatment, while spermidine and spermine concentrations rise following the increase in S-adenosylmethionine decarboxylase. 4. Diaminopropane induces the synthesis of an ornithine decarboxylase-antizyme (mol. wt 19,500 and I, z 12.8 min). A similar antizyme is also induced by diaminobutane, spermidine and spermine. 5. The effect of the antizyme on ornithine decarboxylase is lower in the presence of high concentrations of pvridoxal phosphate. The antizvme loses its activity when treated with pyridoxal phosphate and reduceh-with NaBH,.’
respectively (Grill0 & Bedino, 1977; Grill0 et ul., 1978). Their t,,z is lower than that of the same enzymes in mammals (8 and 22min respectively). Their activity is controlled by ornithine and polyamines, though the administration of these substances does not usually result in a change in the liver polyamine content. The present paper shows that both diaminopropane and natural polyamines inhibit insulin-induced ornithine decarboxylase and S-adenosylmethionine decarboxylase activity in chicken liver partially by triggering the synthesis of an ornithine decarboxylaseantizyme. Some of the properties of this antizyme are also described.
INTRODUCTION
has been reported (Piis & JCnne, 1976) that l,3-diaminopropane, a structural analogue of putrestine (l,Cdiaminobutane), inhibits ornithine decarboxylase stimulation in regenerating rat liver, but has no effect on this enzyme in vitro. Under the same conditions, S-adenosylmethionine decarboxylase activity was increased, while spermidine accumulation was prevented. Therefore these authors suggested that spermidine content in liver depends on ornithine decarboxylase activity only. However later on it has been observed that S-adenosylmethionine decarboxylase is also reduced by administration of diaminopropane (Pbsii et al., 1977). In an attempt to explain the mechanism of ornithine decarboxylase activity regulation by diaminopropane in rat H-35 hepatoma cell cultures, Fong er al. (1976) showed that putrescine elicits the appearance of an inhibitor of ornithine decarboxylase. This inhibitor is a protein and has a molecular weight of 26.500. A similar inhibitor, with a half-life of 24min, has also been demonstrated in rat liver (Heller et al., 1976) and in HTC cell cultures (McCann et al., 1977). According to Kallio et al. (1977) however, diamines may be supposed to regulate ornithine decarboxylase activity in regenerating rat liver by a direct effect on its rate of synthesis at some post-transcriptional level, while the antizyme is not involved in the inhibition of ornithine decarboxylase after a single injection of diamines. The absence of this antizyme in kidney extract, where enzyme activity is completely inhibited by diaminopropane, would support this hypothesis (Pegg et al.. 1978). We have been investigating the regulation of polyamine synthesis in chicken liver for some time and have shown that both ornithine decarboxylase and S-adenosylmethionine decarboxylase are induced by insulin, with a 90-fold and l&fold increase in activity It
II< III
<
MATERIALS AND METHODS
Animals Two to three-week old, I6 hr fasted, male chickens were used. Six hr before sacrifice, one group of animals received i.m. 4.8 i.u. insulin/l00 g body weight, followed by 75 pmols diaminopropane/lOOg i.p. 5 hr to I5 min before sacrifice. Another group received diaminopropane only. Other groups received i.p. 75 pmols diaminobutane. spermidine or spermine, or ornithine plus diaminopropane. To measure the apparent half-life of the ornithine decarboxylase-antizyme, this was induced with diaminopropane. One hr later, I mg cycloheximide/lOO g was injected and the animals were killed after 0, 5, 10, 15 and 20 min.
Assays For ornithine decarboxylase, S-adenosylmethionine decarboxylase and ornithine decarboxylase-antizyme assays. the liver was homogenized just after sacrifice with 4 vol of 0. I5 M KCI. The homogenate was centrifuged for IO min at 12,000 9 and activity was measured in the supernatant. Ornithine decarboxylase activity was measured according to Jlnne & Williams-Ashman (1971). The reaction mixture contained 50 Hmols glycylglycine at pH 7.2, 2.5 pmols dithiothreitol, 0.1 pmols pyridoxal phosphiite. 0.25 pmols 37
38
M. A. GRILLO, S. BEUINO and G. T~STORE
L-ornithine, 0.20 $i D.L-ornithine-I-“‘C and enzyme preparation in 0.5 ml total volume. Incubation time was 60 min. at 37°C in a shaking incubator. “COL was trapped in 0.1 ml of NCS (Nuclear Chicago Solubilizer) contained in plastic centre wells. The reaction was stopped by injecting 0.5 ml of 40”:, trichloroacetic acid and the incubation was continued fo; an additional 20min. The centre well was then removed and droooed into IOml of a PPO-POPOP in toluene solution to c%mt ‘YO,. S-adenosylmethionine decarboxylase activity was measured as described by Sturman (1976). The reaction mixture. containing IO pmols sodium phosphate buffer, pH 7.2. 0.25 pmols putrescine. 0.025 pmols (250,COO dis/min) S-adenosylmethionine and tissue extract in a total volume of 0.1 ml, was incubated at 37’C in a shaking water bath for 30 min. ‘%02 was trapped in 0.1 ml NCS contained in a plastic centre well. Termination of the reaction and counting were performed as described above. For the ornithine decarboxylase antizyme assay the reaction mixture contained 50pmols glycylglycine at pH 7.2, 2.5 pmols dithiothreitol, 0.1 pmols pyridoxal phosphate, 0.25 pmols r-ornithine, 0.2OpCi L&L-ornithine-I-“+C. approx two units ornithine decarboxylase (0.1 ml) and liver extract (usually 0.012 ml) in 0.5 ml total vol. The amount of inhibitor was such that the range of inhibition was never greater than 50%. Incubation time was 60 min at 37°C in a “‘CO2 was trapped and counted as deshaking incubator. scribed above. One unit of inhibitor is the amount that will inhibit one unit of enzyme activity. One unit of enzyme activity catalyzes the release of 1 nmol of CO2 in I hr. Diaminopropuue
and polyamine
assq
Diaminopropane and polyamines were extracted as previously described (Grill0 rf al., 1978) and determined by the method of Tabor et a/. (1973) slightly modified. Analysis was carried out on 0.6ml of the extract with a Jeol model 5AH amino acid analyzer. The diluted extract was applied on the 140 x 12 mm column equilibrated with 0.12 M sodium citrate buffer, pH 4.70, and washed with the same buffer for 15 min. Diaminopropane and polyamines were eluted at 60°C at a flow rate of 1.02 ml/min with 0.12 M sodium citrate butler, pH 5.26. containing I.5 M NaCl; after 98 min the NaCl concentration was increased to 2 M in order to accelerate the elution of spermine. Diaminopentane (0.025 mM) was used as internal standard. The elution times were: diaminopropane 46 min. diaminobutane 53 min. diaminopentane 72 min. spermidine 97 min and spermine 172 min. After each analysis the column was washed for 1hr with 0.2 M NaOH containing O.l”i EDTA. Molecular
weight
determinatiot~
The molecular weight of the antizyme was determined by Bio Gel P-100 (Bio Rad Laboratories) filtration. The proteins contained in the crude extract from chicken liver were precipitated with ammonium sulfate at 50% saturation and centrifuged at 12,000 g for 1Omin. The pellet was then collected in 30mM phosphate buffer, pH 7.2, containing 250 mM NaCl. dialyzed against the same buffer and centrifuged to discard the undissolved material. Three ml of the supernatant (containing 40 units of antizyme) were applied to a 1.4 x 86 cm Bio Gel P-100 column equilibrated with 30mM phosphate buffer, pH 7.2, containing 250mM NaCI, and eluted with the same buffer at a flow rate of 7 ml/‘hr; 2 ml fractions were collected. The column had been previously standardized by eluting 2 ml of a solution containing 10 mg each of bovine serum albumin, carbonic anhydrase from bovine erythrocytes and cytochrome c from horse heart. Chemicals
Ornithine-I-‘% and S-adenosyl-[ I-‘4C]methionine were obtained from New England Nuclear Co.; insulin. cycloheximide. putrescine. spermidine. spermine. diaminopen-
tane, S-adenosylmethionine. serum albumin. carbonic anhydrase, cytochrome c. dithiothreitol. pyridoxal phosphate were Sigma products; diaminopropane was obtained from Fluka.
RESULTS E&t
ofdiuminopropune
ctrld polyamirres
Administration (i.p.) of 7.5 pmols diaminopropane, IOOg in chickens after induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase with insulin promotes fast inhibition of both enzymes: ornithine decarboxylase activity is already decreased by 757; after I5 min and its lowest activity is observed after one hour; the decrease in S-adenosylmethionine decarboxylase activity is observed after 30min. The effect of the diamine does not last long: 5 hr after diaminopropane administration, ornithine decarboxylase values returned to those observed in chickens treated with insulin only; S-adenosylmethionine decarboxylase on the other hand is more active than in the controls, as previously observed in rat liver by P&ii & Jlnne (I 976) (Fig. I ). On measuring diamine and polyamine content in liver, a good correlation is observed between diaminopropane concentration and the decrease of ornithine decarboxylase activity in the first hour; after another 2 hr. however, diaminopropane is again low. while ornithine decarboxylase activity is still at a minimum, No variation in diaminobutane concentration accompanied the decrease in ornithine decarboxylase activity (Fig. 2). Spermidine and spermine concentrations, however, were increased by IS?< 5 hr after treatment with diaminopropane, when ornithine decarboxylase was again normal and S-adenosylmethionine decarboxylase activity was increased (Table I). In vitro, diaminopropane is a weak inhibitor of ornithine decarboxylase activity: with 55 mM diaminopropane there is only 25% inhibition (Table 2). To explain the mechanism of action of diaminopropane, liver enzyme activity has been measured in the presence of the extract from animals in which insulininduction had been blocked by diaminopropane: while S-adenosylmethionine decarboxylase displayed the sum of the two activities, ornithine decarboxylase activity was markedly inhibited. Inhibition was observed even after dialysis of the preparation, but not after heating for 3 min at 100°C (Table 3). The inhibitor precipitates on addition of ammonium sulfate at 50% saturation. This indicates that an inhibitor of ornithine decarboxylase is present in the liver preparation of a diaminopropane treated chicken. This is a protein and can be defined as ornithine decarboxylase-antizyme. To show whether the effect of diamipropane on ornithine decarboxylase is due to induction of an antizyme, ornithine decarboxylase-antizyme activity was measured at different times in the liver of chickens treated with 75 pmols diaminopropane (Fig. 3). The inhibitor was already present in small amounts in normal animals and its concentration increased rapidly to a maximum I hr after diaminopropane. After a further 2 hr, its concentration was again the same as that observed in normal animals. A similar antizyme was also induced by diaminobutane, spermidine and spermine. Insulin had no
39
Induction of ornithine decarboxylase antizyme
Tii of octton of diaminopropona,hr Fig. 1. Effect of diaminopropane on ornithine decarboxylase and S-adenosylmethionine decarboxylase in insulin-treated chickens. Chickens received i.m. 4.8 i.u. insulin/lObg body weight. 15 min to 5 hr before killing, the animals also received 75 pmols diaminopropane/lOO g. Six hr after injection of insulin the animals were killed and liver was used for measuring enzyme activity. Each point represents the mean of at least three determinations. 0-0, ornithine decarboxylase activity (ODC); M, S-adenosylmethionine decarboxylase activity (SAMDC). effect on antizyme ornithine observed Properties
I
I
2
3
4
5
induction, nor was regulation (Table 4).
and localization
by
of the antiryme
Chromatography on Bio Gel P-100 showed that its molecular weight is 19,500 (Fig. 4). Its apparent halflife is 12.8 min (Fig. 5). Antizyme activity was inversely related to pyridoxal phosphate concentration (Table 5). This suggests that the antizyme either binds the enzyme at the same site as the prosthetic group or that it binds the pyridoxal phosphate. When the antizyme is treated with pyridoxal phosphate, reduced with NaBH, and dialyzed, its activity is virtually destroyed (Table 6), so that the second explanation seems more likely. However, the possibility that the antizyme bound to the cofactor is unable to bind the enzyme and to inhibit it cannot be ruled out. The extract separated at 12,000 g was centrifuged for 1 hr at lOO,OOOg to determine the location of the antizyme more precisely. As can be seen in Table 7, most of its activity is in the supernatant (Table 7).
Time of actlon of dlamlnopropane,hr
Fig. 2. Effect of diaminopropane on the concentration of diaminobutane in the liver of insulin treated chickens. Chickens received i.m. 4.8 i.u. insulin/100 g body weight. 15 min to 5 hr before killing, the animals received also 75 pmols diaminopropane/lOO g. Six hr after injection of insulin the animals were killed; the liver rapidly excised was dropped into liquid nitrogen and used for measuring diamines as described under Methods. Each point represents the mean of 3-7 experiments. O---O, diaminopropane; M, diaminobutane.
DISCUSStON Diaminopropane prevents most of ‘ihe increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase induced by insulin in chicken liver. The effect is very fast for ornithine decarboxylase, which is reduced by 75% in 15 min, slower for S-adenosylmethionine decarboxylase, probably due to its longer half-life (22 min, Grill0 et al., 1978) as opposed to
M. A. GRILLO. S. BEDINO and G. TESTORE
40 Table
I. Effect of diaminopropane on spermidine tration in the liver of insulin-treated
and spermine chickens
nmol/g Treatment Insulin Insulin
+ diaminopropane
concen-
liver
Spermidine
Spermine
548.5 * 38.4(3) 640.2 + 9.4 (4)
878.1 + 67.7(4) 1003.5 f 34.4(4)
Chickens received i.m. 4.8i.u. insulin/lOOg body weight. One hour later one group of animals received also 75 pmols diaminopropane/lOO g. Six hours after injection of insulin the animals were killed and the liver rapidly excised and dropped into liquid nitrogen and used for measuring polyamines concentration as described under Methods. Table
2. Effect of diaminopropane in oirro on ornithine decarboxylase activity
Diaminopropane mM
Activity (pm01 CO&r) 726 675 630 548 292
0 II 22 55 110
Inhibition (7;;)
7 13 25 60
Ornithine decarboxylase activity was measured as described under Methods, in the presence and in the absence of different concentrations of diaminopropane.
8 min for ornithine
decarboxylase (Grillo & Bedino. 1977). Liver diaminopropane in the first hour is inversely proportional to ornithine decarboxylase activity, though this remains low after diaminopropane disappears from the liver, suggesting that diaminopropane does not act directly on enzyme activity. Experiments in vitro. showing that much higher diaminopropane concentrations than that present in cytosol (the whole liver concentration is no more than 2 mM) are required to obtain distinct inhibition, support this hypothesis. Moreover, this effect of diaminopropane is not reflected in the diaminobutane content. Only spermidine and spermine are slightly increased 5 hr after administration of the diamine, just as S-adenosylmethionine decarboxylase is elevated. A factor inhibiting ornithine decarboxylase is present in the liver of the diaminopropane-treated chicken. Being heat-labile, not dialyzable, it is thereTable
L
I
2
4
3
Time of action of dlamlnopropone,hr
Fig. 3. Induction of an ornithine decarboxylase antizyme by diaminopropane. The animals received 75pmol diaminopropane/lOOg. After the chosen time as indicated the liver was used for measuring ornithine decarboxylaseantizyme as described under Methods. Each point represents the mean of at least three determinations.
fore a protein, and can be classified as an ornithine decarboxylase-antizyme. A similar compound has been demonstrated in rat hepatoma cell cultures (Heller et al., 1976; McCann et al., 1977) and in rat
3. Effect of the supernatant of a liver homogenate ornithine and S-adenosylmethionine decarboxylase
of diaminopropane-treated activity of insulin-treated
chicken chickens
on liver
Activity (nmols CO&) Ornithine decarboxylase Liver supernatant of diaminopropane-treated chicken Ornithine decarboxylase + liver supernatant Ornithine decarboxylase + dialyzed liver supernatant Ornithine decarboxylase + liver supernatant kept 3’ at 100°C S-adenosylmethionine decarboxylase, insulin-treated Liver supernatant of diaminopropane-treated chicken Liver supernatant of diaminopropane + insulin-treated
2.52 0.00 0.64 0.84 2.30 0.37 0.13 0.50
Ornithine and S-adenosylmethionine decarboxylase activities were measured in the absence and in the presence of 0.1 ml of the supernatant separated at 12,000 g of a 20% liver homogenate in 0.15 M KC1 of diaminopropane-treated chicken (75 ~mols/lOO g body weight, for 1 hr). Where indicated, supernatant was dialyzed overnight against 0.15 M KCI or kept 3 min in a boiling water bath and centrifuged.
Induction
of ornithine
decarboxylase
41
antizyme
Table 4. Induction of an ornithine decarboxylase-antizyme by diaminopropane, diaminobutane, spermidine and spermine in chicken liver. Effect of insulin and of ornithine Units antizyme/g Controls + diaminopropane + diaminobutane + spermidine + spermine + diaminopropane + diaminopropane
27 196 117 175 95 165 207
+ insulin + ornithine
f f * + f + +
liver
6 (3) 23 (6) 30(4) 15(3) 18(6) 28 (5) 18(5)
Groups of chickens received i.p. 75pmols either diaminopropane or diaminobutane or spermidine or spermine. Other groups received i.m. 4.8 i.u. insulin and i.p. 75 pmols diaminopropane and respectively 75 ymols each diaminopropane and ornithine. One hour after the injections, animals were killed and ornithine decarboxylase-antizyme was measured as described under Methods.
70 c
I
2
5
3
IO IO’ 5
Molecular
liver (Heller et al., 1976; Heller et al., 19?7). t,,, of the ornithine decarboxylase-antizyme of chicken liver is 12.8 min. while that of the enzyme is 8 min. tljs of the chicken liver antizyme is therefore low when com-
phosphate mM 0.2 0.2 2.5 4.0
20
n-t”
Fig. 5. Apparent half-life of ornithine decarboxylase-antizyme of chicken liver. One hr after injection of diaminopropane, the chickens received i.p. I mg cycloheximide/ 100 g. Antizyme activity was assayed at the time indicated. The line was plotted with the least-squares method.
pared with the 18-66 min reported by Heller et al. (1976) in rat liver and rat hepatoma cells. The molecular weight (19,500) of the antizyme is somewhat less than the 26,500 observed in mammals. A similar ornithine decarboxylase-antizyme is also induced by diaminobutane, spermidine and spermine.
5. Effect of pyridoxal phosphate on the inhibition decarboxylase by ornithine decarboxylase-antizyme
Pyridoxal
15 Time,
Fig. 4. Molecular weight determination of ornithine decarboxylase-antizyme of chicken liver. Approx 40 units of inhibitor were applied to a 1.4 x 86cm Bio Gel P-100 column previously standardized with serum albumin (67,000), carbonic anhydrase (30,000) and cytochrome c (I 3,370). Antizyme was measured on 0.3 ml of each fraction. A, cytochrome c; B, carbonic anhydrase; C, serum albumin; 0, antizyme.
Table
IO
welght
Ornithine decarboxylase activity (units)
of ornithine
Inhibition (“/,)
4.01’ 2.17 2.63 2.87
* Reference activity measured in the absence of antizyme. Ornithine decarboxylase-antizyme was assayed as described Methods, using three pyridoxal phosphate concentrations.
46 34 28
under
M. A. GRILLO,S. BEDINOand G. TEST~RE
42
Table 6. Effect of treatment with pyridoxal phosphate, followed by reduction, with NaBH,, on the activity of ornithine decarboxylase-antizyme of chicken liver Inhibitor
Units of antizyme
12,000 g supernatant 12,000 g supernatant treated with pyridoxal phosphate and reduced
0.30 0.03
Approx 30 units of inhibitor in 2 ml were treated with 1ml of 2 mM pyridoxal phosphate; after few min. it was reduced with excess borohydride and dialyzed overnight against 30 mM phosphate buffer, pH 7.0. Antizyme was measured as described under Methods. Table 7. Localization of ornithine decarboxylase-antizyme in chicken liver
CRILLO M. A. & BEDINOS. (1977) Stimulation of chicken liver ornithine decarboxylase. Int. J. Biochem. 8, 711-713.
Units of antizyme 0.99 0.88 0.22
12,000 g supernatant lOfJ,tXKJ g supernatant Microsomes
The 12,OOOg supernatant was centrifuged 60 min at 100,000g. The pellet was resuspended in 0.15 M KC1 and taken to the initial volume. The inhibition activity was measured in all fractions as described under Methods.
GRILU) M. A., BEDINOS. & TE~TOREG. (1978) Regulation of polyamine synthesis in chicken liver. Inc. J. Biochem. 9, 185-189. HELLERJ. S., FONG W. F. & CANELLAKISE. S. (1976) Induction of a protein inhibitor to ornithine decarboxvlase by the end products of its reaction. Proc. natn. Acad.‘Sci,, U.S.A. 73, 1858-1862. HELLERJ. S., KYRIAKIDISD., FONG W. F., CANELLAKIS E. S. (1977) Ornithine decarboxylase antizyme is a normal component of uninduced H-35 cells and rat liver. Eur. J. Biochem.
though its value at 1 hr is much less than after diaminopropane. Since it is already present in small amounts in normal liver and is rapidly induced by diamines and polyamines, this ornithine decarboxylase-antizyme may help to regulate ornithine decarboxylase activity, contrary to the observations of Pegg et al. (1978) in rat liver. Its half-life, however, is too short to explain the proIonged inhibition of ornithine decarboxylase activity. Diaminopropane may thus be supposed to act via a further mechanism. It may, for example, affect protein synthesis, as suggested by Kallio et al. (1977) as an explanation for their findings in regenerating rat liver. This may also be true for S-adenosylmethionine decarboxylase, which was not inhibited by preparations in which the antizyme had been induced. The experimental evidence indicates that the ornithine decarboxylase antizyme binds with pyridoxal phosphate, i.e. the prosthetic group of the enzyme. This means that it probably has an amino group at the active site, REFERENCES FONG W. S., HELLERJ. S. & CANELLAKIS E. S. (1976) The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures Biochim. biophys. Acta 428. 45&465.
81. 545-550.
JLNNEJ., WILLIAMS-ASHMAN H. G. (1971) On the purification of r_-ornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme. J. biol. Chem. 246, 1725-1732. KALLIOA., L~FMANM., P0sd H. & JKNNEJ. (1977) Inhibition of ornithine decarboxylase by diamines in regenerating rat liver. Evidence for direct action on the accumulation of the enzyme protein. FEBS Lett. 79, 195-199. MCCANN P. P., TARDIFC. & MAMONTP. S. (1977) x I Reeula-a---tion of ornithine decarboxylase by ODC-antizyme in HTC cells. Biochem. biophys. Rex Commun. 75, 948-954. PEGG A. E., CONOVERC. & WRONAA. (1978) Effects of aliphatic diamines on rat liver ornithine decarboxylase activity. Biochem. J. 170, 651-660. P&0 H., KALLIO A., SCALABRINO G. & JKNNE J. (1977) Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in uico. Biochim.
biophys.
Acta 497, 288-297.
Pi%0 H. & JKNNEJ. (1976) Inhibition of ornithine decarboxylase activity and spermidine accumulation in regenerating rat liver. 69, 885-892. STURMANJ. A. (1976) Subcellular distribution of S-adenosylmethionine decarboxylase in rat liver. Evidence of decarboxylation of S-adenosylmethionine separate from synthesis of spermidine. Biochim. biophys. Acta 428, 5&69.
TABORH., TABORC. W. & IRREVERRE F. (1973) Quantitative determination of aliphatic diamines and polyamines by an automated liquid chromatography procedure. Analyt.
Biochem.
55, 457467.
INDUCTION OF AN ORNITHINE DECARBOXYLASE-ANTIZYME IN CHICKEN M. A. Cattedra
di Chimica
GRILLO,
S. BEDINO
LIVER
and G. TESTORE
e Istituto di Chimica biologica della Facolta dell’Universit8 di Torino, Torino, Italy
di Medicina
e Chirurgia
(Rrceived 18 May 1979) Abstract---l. The insulin-induced increase in ornithine decarboxylase and in S-adenosylmethionine decarboxylase in chicken liver is considerably inhibited by diaminopropane. This effect does not last long: after 5 hr, ornithine decarboxylase has returned to normal, though S-adenosylmethionine decarboxylase is more active. 2. There is a good correlation between diaminopropane and ornithine decarboxylase activity for I hr. after which ornithine decarboxylase remains low, even when diaminopropane disappears from the liver. 3. Liver diaminobutane concentration is fairly constant throughout the treatment, while spermidine and spermine concentrations rise following the increase in S-adenosylmethionine decarboxylase. 4. Diaminopropane induces the synthesis of an ornithine decarboxylase-antizyme (mol. wt 19,500 and I, z 12.8 min). A similar antizyme is also induced by diaminobutane, spermidine and spermine. 5. The effect of the antizyme on ornithine decarboxylase is lower in the presence of high concentrations of pvridoxal phosphate. The antizvme loses its activity when treated with pyridoxal phosphate and reduceh-with NaBH,.’
respectively (Grill0 & Bedino, 1977; Grill0 et ul., 1978). Their t,,z is lower than that of the same enzymes in mammals (8 and 22min respectively). Their activity is controlled by ornithine and polyamines, though the administration of these substances does not usually result in a change in the liver polyamine content. The present paper shows that both diaminopropane and natural polyamines inhibit insulin-induced ornithine decarboxylase and S-adenosylmethionine decarboxylase activity in chicken liver partially by triggering the synthesis of an ornithine decarboxylaseantizyme. Some of the properties of this antizyme are also described.
INTRODUCTION
has been reported (Piis & JCnne, 1976) that l,3-diaminopropane, a structural analogue of putrestine (l,Cdiaminobutane), inhibits ornithine decarboxylase stimulation in regenerating rat liver, but has no effect on this enzyme in vitro. Under the same conditions, S-adenosylmethionine decarboxylase activity was increased, while spermidine accumulation was prevented. Therefore these authors suggested that spermidine content in liver depends on ornithine decarboxylase activity only. However later on it has been observed that S-adenosylmethionine decarboxylase is also reduced by administration of diaminopropane (Pbsii et al., 1977). In an attempt to explain the mechanism of ornithine decarboxylase activity regulation by diaminopropane in rat H-35 hepatoma cell cultures, Fong er al. (1976) showed that putrescine elicits the appearance of an inhibitor of ornithine decarboxylase. This inhibitor is a protein and has a molecular weight of 26.500. A similar inhibitor, with a half-life of 24min, has also been demonstrated in rat liver (Heller et al., 1976) and in HTC cell cultures (McCann et al., 1977). According to Kallio et al. (1977) however, diamines may be supposed to regulate ornithine decarboxylase activity in regenerating rat liver by a direct effect on its rate of synthesis at some post-transcriptional level, while the antizyme is not involved in the inhibition of ornithine decarboxylase after a single injection of diamines. The absence of this antizyme in kidney extract, where enzyme activity is completely inhibited by diaminopropane, would support this hypothesis (Pegg et al.. 1978). We have been investigating the regulation of polyamine synthesis in chicken liver for some time and have shown that both ornithine decarboxylase and S-adenosylmethionine decarboxylase are induced by insulin, with a 90-fold and l&fold increase in activity It
II< III
<
MATERIALS AND METHODS
Animals Two to three-week old, I6 hr fasted, male chickens were used. Six hr before sacrifice, one group of animals received i.m. 4.8 i.u. insulin/l00 g body weight, followed by 75 pmols diaminopropane/lOOg i.p. 5 hr to I5 min before sacrifice. Another group received diaminopropane only. Other groups received i.p. 75 pmols diaminobutane. spermidine or spermine, or ornithine plus diaminopropane. To measure the apparent half-life of the ornithine decarboxylase-antizyme, this was induced with diaminopropane. One hr later, I mg cycloheximide/lOO g was injected and the animals were killed after 0, 5, 10, 15 and 20 min.
Assays For ornithine decarboxylase, S-adenosylmethionine decarboxylase and ornithine decarboxylase-antizyme assays. the liver was homogenized just after sacrifice with 4 vol of 0. I5 M KCI. The homogenate was centrifuged for IO min at 12,000 9 and activity was measured in the supernatant. Ornithine decarboxylase activity was measured according to Jlnne & Williams-Ashman (1971). The reaction mixture contained 50 Hmols glycylglycine at pH 7.2, 2.5 pmols dithiothreitol, 0.1 pmols pyridoxal phosphiite. 0.25 pmols 37
38
M. A. GRILLO, S. BEUINO and G. T~STORE
L-ornithine, 0.20 $i D.L-ornithine-I-“‘C and enzyme preparation in 0.5 ml total volume. Incubation time was 60 min. at 37°C in a shaking incubator. “COL was trapped in 0.1 ml of NCS (Nuclear Chicago Solubilizer) contained in plastic centre wells. The reaction was stopped by injecting 0.5 ml of 40”:, trichloroacetic acid and the incubation was continued fo; an additional 20min. The centre well was then removed and droooed into IOml of a PPO-POPOP in toluene solution to c%mt ‘YO,. S-adenosylmethionine decarboxylase activity was measured as described by Sturman (1976). The reaction mixture. containing IO pmols sodium phosphate buffer, pH 7.2. 0.25 pmols putrescine. 0.025 pmols (250,COO dis/min) S-adenosylmethionine and tissue extract in a total volume of 0.1 ml, was incubated at 37’C in a shaking water bath for 30 min. ‘%02 was trapped in 0.1 ml NCS contained in a plastic centre well. Termination of the reaction and counting were performed as described above. For the ornithine decarboxylase antizyme assay the reaction mixture contained 50pmols glycylglycine at pH 7.2, 2.5 pmols dithiothreitol, 0.1 pmols pyridoxal phosphate, 0.25 pmols r-ornithine, 0.2OpCi L&L-ornithine-I-“+C. approx two units ornithine decarboxylase (0.1 ml) and liver extract (usually 0.012 ml) in 0.5 ml total vol. The amount of inhibitor was such that the range of inhibition was never greater than 50%. Incubation time was 60 min at 37°C in a “‘CO2 was trapped and counted as deshaking incubator. scribed above. One unit of inhibitor is the amount that will inhibit one unit of enzyme activity. One unit of enzyme activity catalyzes the release of 1 nmol of CO2 in I hr. Diaminopropuue
and polyamine
assq
Diaminopropane and polyamines were extracted as previously described (Grill0 rf al., 1978) and determined by the method of Tabor et a/. (1973) slightly modified. Analysis was carried out on 0.6ml of the extract with a Jeol model 5AH amino acid analyzer. The diluted extract was applied on the 140 x 12 mm column equilibrated with 0.12 M sodium citrate buffer, pH 4.70, and washed with the same buffer for 15 min. Diaminopropane and polyamines were eluted at 60°C at a flow rate of 1.02 ml/min with 0.12 M sodium citrate butler, pH 5.26. containing I.5 M NaCl; after 98 min the NaCl concentration was increased to 2 M in order to accelerate the elution of spermine. Diaminopentane (0.025 mM) was used as internal standard. The elution times were: diaminopropane 46 min. diaminobutane 53 min. diaminopentane 72 min. spermidine 97 min and spermine 172 min. After each analysis the column was washed for 1hr with 0.2 M NaOH containing O.l”i EDTA. Molecular
weight
determinatiot~
The molecular weight of the antizyme was determined by Bio Gel P-100 (Bio Rad Laboratories) filtration. The proteins contained in the crude extract from chicken liver were precipitated with ammonium sulfate at 50% saturation and centrifuged at 12,000 g for 1Omin. The pellet was then collected in 30mM phosphate buffer, pH 7.2, containing 250 mM NaCl. dialyzed against the same buffer and centrifuged to discard the undissolved material. Three ml of the supernatant (containing 40 units of antizyme) were applied to a 1.4 x 86 cm Bio Gel P-100 column equilibrated with 30mM phosphate buffer, pH 7.2, containing 250mM NaCI, and eluted with the same buffer at a flow rate of 7 ml/‘hr; 2 ml fractions were collected. The column had been previously standardized by eluting 2 ml of a solution containing 10 mg each of bovine serum albumin, carbonic anhydrase from bovine erythrocytes and cytochrome c from horse heart. Chemicals
Ornithine-I-‘% and S-adenosyl-[ I-‘4C]methionine were obtained from New England Nuclear Co.; insulin. cycloheximide. putrescine. spermidine. spermine. diaminopen-
tane, S-adenosylmethionine. serum albumin. carbonic anhydrase, cytochrome c. dithiothreitol. pyridoxal phosphate were Sigma products; diaminopropane was obtained from Fluka.
RESULTS E&t
ofdiuminopropune
ctrld polyamirres
Administration (i.p.) of 7.5 pmols diaminopropane, IOOg in chickens after induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase with insulin promotes fast inhibition of both enzymes: ornithine decarboxylase activity is already decreased by 757; after I5 min and its lowest activity is observed after one hour; the decrease in S-adenosylmethionine decarboxylase activity is observed after 30min. The effect of the diamine does not last long: 5 hr after diaminopropane administration, ornithine decarboxylase values returned to those observed in chickens treated with insulin only; S-adenosylmethionine decarboxylase on the other hand is more active than in the controls, as previously observed in rat liver by P&ii & Jlnne (I 976) (Fig. I ). On measuring diamine and polyamine content in liver, a good correlation is observed between diaminopropane concentration and the decrease of ornithine decarboxylase activity in the first hour; after another 2 hr. however, diaminopropane is again low. while ornithine decarboxylase activity is still at a minimum, No variation in diaminobutane concentration accompanied the decrease in ornithine decarboxylase activity (Fig. 2). Spermidine and spermine concentrations, however, were increased by IS?< 5 hr after treatment with diaminopropane, when ornithine decarboxylase was again normal and S-adenosylmethionine decarboxylase activity was increased (Table I). In vitro, diaminopropane is a weak inhibitor of ornithine decarboxylase activity: with 55 mM diaminopropane there is only 25% inhibition (Table 2). To explain the mechanism of action of diaminopropane, liver enzyme activity has been measured in the presence of the extract from animals in which insulininduction had been blocked by diaminopropane: while S-adenosylmethionine decarboxylase displayed the sum of the two activities, ornithine decarboxylase activity was markedly inhibited. Inhibition was observed even after dialysis of the preparation, but not after heating for 3 min at 100°C (Table 3). The inhibitor precipitates on addition of ammonium sulfate at 50% saturation. This indicates that an inhibitor of ornithine decarboxylase is present in the liver preparation of a diaminopropane treated chicken. This is a protein and can be defined as ornithine decarboxylase-antizyme. To show whether the effect of diamipropane on ornithine decarboxylase is due to induction of an antizyme, ornithine decarboxylase-antizyme activity was measured at different times in the liver of chickens treated with 75 pmols diaminopropane (Fig. 3). The inhibitor was already present in small amounts in normal animals and its concentration increased rapidly to a maximum I hr after diaminopropane. After a further 2 hr, its concentration was again the same as that observed in normal animals. A similar antizyme was also induced by diaminobutane, spermidine and spermine. Insulin had no
39
Induction of ornithine decarboxylase antizyme
Tii of octton of diaminopropona,hr Fig. 1. Effect of diaminopropane on ornithine decarboxylase and S-adenosylmethionine decarboxylase in insulin-treated chickens. Chickens received i.m. 4.8 i.u. insulin/lObg body weight. 15 min to 5 hr before killing, the animals also received 75 pmols diaminopropane/lOO g. Six hr after injection of insulin the animals were killed and liver was used for measuring enzyme activity. Each point represents the mean of at least three determinations. 0-0, ornithine decarboxylase activity (ODC); M, S-adenosylmethionine decarboxylase activity (SAMDC). effect on antizyme ornithine observed Properties
I
I
2
3
4
5
induction, nor was regulation (Table 4).
and localization
by
of the antiryme
Chromatography on Bio Gel P-100 showed that its molecular weight is 19,500 (Fig. 4). Its apparent halflife is 12.8 min (Fig. 5). Antizyme activity was inversely related to pyridoxal phosphate concentration (Table 5). This suggests that the antizyme either binds the enzyme at the same site as the prosthetic group or that it binds the pyridoxal phosphate. When the antizyme is treated with pyridoxal phosphate, reduced with NaBH, and dialyzed, its activity is virtually destroyed (Table 6), so that the second explanation seems more likely. However, the possibility that the antizyme bound to the cofactor is unable to bind the enzyme and to inhibit it cannot be ruled out. The extract separated at 12,000 g was centrifuged for 1 hr at lOO,OOOg to determine the location of the antizyme more precisely. As can be seen in Table 7, most of its activity is in the supernatant (Table 7).
Time of actlon of dlamlnopropane,hr
Fig. 2. Effect of diaminopropane on the concentration of diaminobutane in the liver of insulin treated chickens. Chickens received i.m. 4.8 i.u. insulin/100 g body weight. 15 min to 5 hr before killing, the animals received also 75 pmols diaminopropane/lOO g. Six hr after injection of insulin the animals were killed; the liver rapidly excised was dropped into liquid nitrogen and used for measuring diamines as described under Methods. Each point represents the mean of 3-7 experiments. O---O, diaminopropane; M, diaminobutane.
DISCUSStON Diaminopropane prevents most of ‘ihe increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase induced by insulin in chicken liver. The effect is very fast for ornithine decarboxylase, which is reduced by 75% in 15 min, slower for S-adenosylmethionine decarboxylase, probably due to its longer half-life (22 min, Grill0 et al., 1978) as opposed to
M. A. GRILLO. S. BEDINO and G. TESTORE
40 Table
I. Effect of diaminopropane on spermidine tration in the liver of insulin-treated
and spermine chickens
nmol/g Treatment Insulin Insulin
+ diaminopropane
concen-
liver
Spermidine
Spermine
548.5 * 38.4(3) 640.2 + 9.4 (4)
878.1 + 67.7(4) 1003.5 f 34.4(4)
Chickens received i.m. 4.8i.u. insulin/lOOg body weight. One hour later one group of animals received also 75 pmols diaminopropane/lOO g. Six hours after injection of insulin the animals were killed and the liver rapidly excised and dropped into liquid nitrogen and used for measuring polyamines concentration as described under Methods. Table
2. Effect of diaminopropane in oirro on ornithine decarboxylase activity
Diaminopropane mM
Activity (pm01 CO&r) 726 675 630 548 292
0 II 22 55 110
Inhibition (7;;)
7 13 25 60
Ornithine decarboxylase activity was measured as described under Methods, in the presence and in the absence of different concentrations of diaminopropane.
8 min for ornithine
decarboxylase (Grillo & Bedino. 1977). Liver diaminopropane in the first hour is inversely proportional to ornithine decarboxylase activity, though this remains low after diaminopropane disappears from the liver, suggesting that diaminopropane does not act directly on enzyme activity. Experiments in vitro. showing that much higher diaminopropane concentrations than that present in cytosol (the whole liver concentration is no more than 2 mM) are required to obtain distinct inhibition, support this hypothesis. Moreover, this effect of diaminopropane is not reflected in the diaminobutane content. Only spermidine and spermine are slightly increased 5 hr after administration of the diamine, just as S-adenosylmethionine decarboxylase is elevated. A factor inhibiting ornithine decarboxylase is present in the liver of the diaminopropane-treated chicken. Being heat-labile, not dialyzable, it is thereTable
L
I
2
4
3
Time of action of dlamlnopropone,hr
Fig. 3. Induction of an ornithine decarboxylase antizyme by diaminopropane. The animals received 75pmol diaminopropane/lOOg. After the chosen time as indicated the liver was used for measuring ornithine decarboxylaseantizyme as described under Methods. Each point represents the mean of at least three determinations.
fore a protein, and can be classified as an ornithine decarboxylase-antizyme. A similar compound has been demonstrated in rat hepatoma cell cultures (Heller et al., 1976; McCann et al., 1977) and in rat
3. Effect of the supernatant of a liver homogenate ornithine and S-adenosylmethionine decarboxylase
of diaminopropane-treated activity of insulin-treated
chicken chickens
on liver
Activity (nmols CO&) Ornithine decarboxylase Liver supernatant of diaminopropane-treated chicken Ornithine decarboxylase + liver supernatant Ornithine decarboxylase + dialyzed liver supernatant Ornithine decarboxylase + liver supernatant kept 3’ at 100°C S-adenosylmethionine decarboxylase, insulin-treated Liver supernatant of diaminopropane-treated chicken Liver supernatant of diaminopropane + insulin-treated
2.52 0.00 0.64 0.84 2.30 0.37 0.13 0.50
Ornithine and S-adenosylmethionine decarboxylase activities were measured in the absence and in the presence of 0.1 ml of the supernatant separated at 12,000 g of a 20% liver homogenate in 0.15 M KC1 of diaminopropane-treated chicken (75 ~mols/lOO g body weight, for 1 hr). Where indicated, supernatant was dialyzed overnight against 0.15 M KCI or kept 3 min in a boiling water bath and centrifuged.
Induction
of ornithine
decarboxylase
41
antizyme
Table 4. Induction of an ornithine decarboxylase-antizyme by diaminopropane, diaminobutane, spermidine and spermine in chicken liver. Effect of insulin and of ornithine Units antizyme/g Controls + diaminopropane + diaminobutane + spermidine + spermine + diaminopropane + diaminopropane
27 196 117 175 95 165 207
+ insulin + ornithine
f f * + f + +
liver
6 (3) 23 (6) 30(4) 15(3) 18(6) 28 (5) 18(5)
Groups of chickens received i.p. 75pmols either diaminopropane or diaminobutane or spermidine or spermine. Other groups received i.m. 4.8 i.u. insulin and i.p. 75 pmols diaminopropane and respectively 75 ymols each diaminopropane and ornithine. One hour after the injections, animals were killed and ornithine decarboxylase-antizyme was measured as described under Methods.
70 c
I
2
5
3
IO IO’ 5
Molecular
liver (Heller et al., 1976; Heller et al., 19?7). t,,, of the ornithine decarboxylase-antizyme of chicken liver is 12.8 min. while that of the enzyme is 8 min. tljs of the chicken liver antizyme is therefore low when com-
phosphate mM 0.2 0.2 2.5 4.0
20
n-t”
Fig. 5. Apparent half-life of ornithine decarboxylase-antizyme of chicken liver. One hr after injection of diaminopropane, the chickens received i.p. I mg cycloheximide/ 100 g. Antizyme activity was assayed at the time indicated. The line was plotted with the least-squares method.
pared with the 18-66 min reported by Heller et al. (1976) in rat liver and rat hepatoma cells. The molecular weight (19,500) of the antizyme is somewhat less than the 26,500 observed in mammals. A similar ornithine decarboxylase-antizyme is also induced by diaminobutane, spermidine and spermine.
5. Effect of pyridoxal phosphate on the inhibition decarboxylase by ornithine decarboxylase-antizyme
Pyridoxal
15 Time,
Fig. 4. Molecular weight determination of ornithine decarboxylase-antizyme of chicken liver. Approx 40 units of inhibitor were applied to a 1.4 x 86cm Bio Gel P-100 column previously standardized with serum albumin (67,000), carbonic anhydrase (30,000) and cytochrome c (I 3,370). Antizyme was measured on 0.3 ml of each fraction. A, cytochrome c; B, carbonic anhydrase; C, serum albumin; 0, antizyme.
Table
IO
welght
Ornithine decarboxylase activity (units)
of ornithine
Inhibition (“/,)
4.01’ 2.17 2.63 2.87
* Reference activity measured in the absence of antizyme. Ornithine decarboxylase-antizyme was assayed as described Methods, using three pyridoxal phosphate concentrations.
46 34 28
under
M. A. GRILLO,S. BEDINOand G. TEST~RE
42
Table 6. Effect of treatment with pyridoxal phosphate, followed by reduction, with NaBH,, on the activity of ornithine decarboxylase-antizyme of chicken liver Inhibitor
Units of antizyme
12,000 g supernatant 12,000 g supernatant treated with pyridoxal phosphate and reduced
0.30 0.03
Approx 30 units of inhibitor in 2 ml were treated with 1ml of 2 mM pyridoxal phosphate; after few min. it was reduced with excess borohydride and dialyzed overnight against 30 mM phosphate buffer, pH 7.0. Antizyme was measured as described under Methods. Table 7. Localization of ornithine decarboxylase-antizyme in chicken liver
CRILLO M. A. & BEDINOS. (1977) Stimulation of chicken liver ornithine decarboxylase. Int. J. Biochem. 8, 711-713.
Units of antizyme 0.99 0.88 0.22
12,000 g supernatant lOfJ,tXKJ g supernatant Microsomes
The 12,OOOg supernatant was centrifuged 60 min at 100,000g. The pellet was resuspended in 0.15 M KC1 and taken to the initial volume. The inhibition activity was measured in all fractions as described under Methods.
GRILU) M. A., BEDINOS. & TE~TOREG. (1978) Regulation of polyamine synthesis in chicken liver. Inc. J. Biochem. 9, 185-189. HELLERJ. S., FONG W. F. & CANELLAKISE. S. (1976) Induction of a protein inhibitor to ornithine decarboxvlase by the end products of its reaction. Proc. natn. Acad.‘Sci,, U.S.A. 73, 1858-1862. HELLERJ. S., KYRIAKIDISD., FONG W. F., CANELLAKIS E. S. (1977) Ornithine decarboxylase antizyme is a normal component of uninduced H-35 cells and rat liver. Eur. J. Biochem.
though its value at 1 hr is much less than after diaminopropane. Since it is already present in small amounts in normal liver and is rapidly induced by diamines and polyamines, this ornithine decarboxylase-antizyme may help to regulate ornithine decarboxylase activity, contrary to the observations of Pegg et al. (1978) in rat liver. Its half-life, however, is too short to explain the proIonged inhibition of ornithine decarboxylase activity. Diaminopropane may thus be supposed to act via a further mechanism. It may, for example, affect protein synthesis, as suggested by Kallio et al. (1977) as an explanation for their findings in regenerating rat liver. This may also be true for S-adenosylmethionine decarboxylase, which was not inhibited by preparations in which the antizyme had been induced. The experimental evidence indicates that the ornithine decarboxylase antizyme binds with pyridoxal phosphate, i.e. the prosthetic group of the enzyme. This means that it probably has an amino group at the active site, REFERENCES FONG W. S., HELLERJ. S. & CANELLAKIS E. S. (1976) The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures Biochim. biophys. Acta 428. 45&465.
81. 545-550.
JLNNEJ., WILLIAMS-ASHMAN H. G. (1971) On the purification of r_-ornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme. J. biol. Chem. 246, 1725-1732. KALLIOA., L~FMANM., P0sd H. & JKNNEJ. (1977) Inhibition of ornithine decarboxylase by diamines in regenerating rat liver. Evidence for direct action on the accumulation of the enzyme protein. FEBS Lett. 79, 195-199. MCCANN P. P., TARDIFC. & MAMONTP. S. (1977) x I Reeula-a---tion of ornithine decarboxylase by ODC-antizyme in HTC cells. Biochem. biophys. Rex Commun. 75, 948-954. PEGG A. E., CONOVERC. & WRONAA. (1978) Effects of aliphatic diamines on rat liver ornithine decarboxylase activity. Biochem. J. 170, 651-660. P&0 H., KALLIO A., SCALABRINO G. & JKNNE J. (1977) Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in uico. Biochim.
biophys.
Acta 497, 288-297.
Pi%0 H. & JKNNEJ. (1976) Inhibition of ornithine decarboxylase activity and spermidine accumulation in regenerating rat liver. 69, 885-892. STURMANJ. A. (1976) Subcellular distribution of S-adenosylmethionine decarboxylase in rat liver. Evidence of decarboxylation of S-adenosylmethionine separate from synthesis of spermidine. Biochim. biophys. Acta 428, 5&69.
TABORH., TABORC. W. & IRREVERRE F. (1973) Quantitative determination of aliphatic diamines and polyamines by an automated liquid chromatography procedure. Analyt.
Biochem.
55, 457467.