Occurrence and induction of spermidine-N1-acetyltransferase in Escherichia coli

Spermidine acetylase activity was detected in extracts prepared from $\text{Escherichia coli}$ and there was a marked increase in activity over the early period of growth. This increase reached a maximum 3 h after inoculation and was followed by an increase in ornithine decarboxylase activity. The acetylase was also able to use spermine as a substrate, but not putrescine. With spermidine and acetyl-CoA as substrate, the product formed was exclusively N¹-acetyl-spermidine. This is the first evidence for the occurrence in bacteria of spermidine-N¹-acetyltransferase, an enzyme which has previously been described in mammalian cells. These results suggest that acetylation of spermidine may be involved in the growth of $\text{Escherichia coli}$ and in the regulation of its polyamine content.     

Studies of the induction of spermidine/spermine N1-acetyltransferase using a specific antiserum

A specific antiserum to rat liver spermidine/spermine N1-acetyltransferase was used to study the induction of this protein. The antiserum had no effect on the spermidine acetylating capacity of crude nuclear extracts and very little effect on the activity present in crude cytosolic extracts from control rat tissues indicating that most of this activity is not due to spermidine/spermine N1-acetyltransferase. Treatment of rats with carbon tetrachloride, spermidine, thioacetamide, or methylglyoxal bis(guanylhydrazone) produced a substantial increase in the spermidine acetylating capacity of rat liver cytosolic extracts which was exclusively due to an increase in the immunoprecipitable spermidine/spermine N1-acetyltransferase protein. Exact measurement of the extent of this increase was not possible because the basal amount was too low to determine precisely but the amount of this enzyme increased about 250-fold with 6 h of treatment with carbon tetrachloride, about 25-fold at 6 h after spermidine, about 23-fold at 24 h after thioacetamide and up to 300-fold at 24 h after methylglyoxal bis(guanylhydrazone). Treatment of rats with spermidine also increased spermidine/spermine N1-acetyltransferase in other tissues including lung, kidney, and pancreas. The spermidine/spermine N1-acetyltransferase protein was found to turn over very rapidly with a half-life of about 15 min in thioacetamide-treated rats and 180 min after carbon tetrachloride.     

Effect of inhibitors of protein synthesis on rat liver spermidine N-acetyltransferase

The increase in spermidine N-acetyltransferase activity in rat liver produced by carbon tetrachloride was completely prevented by simultaneous treatment with inhibitors of protein and nucleic acid synthesis suggesting that the increase results from the synthesis of new protein rather than the release of the enzyme from a cryptic inactive form. Treatment with cycloheximide 2 h after carbon tetrachloride also completely blocked the rise in spermidine N-acetyltransferase seen 4 h later. Such treatment completely prevented the fall in spermidine and rise in putrescine in the liver 6 h after carbon tetrachloride confirming the importance of the induction of spermidine N-acetyltransferase in the conversion of spermidine into putrescine. When cycloheximide was administered to rats in which spermidine N-acetyltransferase activity had been stimulated by prior treatment with carbon tetrachloride or thioacetamide, the activity was lost rapidly showing that the enzyme protein has a rapid rate of turnover. The half-life for the enzyme in thioacetamide-treated rats was 40 min, whereas the half-life for ornithine decarboxylase (which is well known to turn over very rapidly) was 27 min. In carbon tetrachloride-treated rats the rate or protein degradation was reduced and the half-life of spermidine N-acetyltransferase was 155 min and that for ornithine decarboxylase was 65 min. It appears that three of the enzymes involved in the synthesis and interconversion of putrescine and spermidine namely, ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine N-acetyltransferase have rapid rates of turnover and that polyamine levels are regulated by changes in the amount of these enzymes.     

Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride

A single intraperitoneal injection of carbon tetrachloride into rats resulted within 12 hours in a marked accumulation of putrescine in liver with a concomitant decrease in the concentration of spermidine. The accumulation of putrescine apparently was partly due to an immense stimulation of ornithine decarboxylase activity occurring at the same time. However, in addition it was found that during the maximal accumulation of putrescine there was a marked incorporation of radioactivity from labelled spermidine to liver putrescine $\text{in vivo}$. The conversion of spermidine to liver putrescine was hardly detectable in control animals. Besides the treatment with carbon tetrachloride, increased conversion of radioactive spermidine to liver putrescine $\text{in vivo}$ also occurred after treatment with growth hormone, after partial hepatectomy and after treatment with thioacetamide, $\text{i. e.}$ under circumstances characterized by a stimulation of ornithine decarboxylase activity and an increased accumulation of putrescine.     

Effects of aliphatic diamines on rat liver ornithine decarboxylase activity.

Rat liver ornithine decarboxylase activity was decreased by administration of putrescine (1,4-diaminobutane) or other diamines, including 1,3-diaminopropane, 1,5-diaminopentane and 1,6-diaminohexane. This effect was seen in control rats and in rats in which hepatic ornithine decarboxylase activity had been increased by administration of growth hormone (somatotropin) or thioacetamide. Loss of activity was not dependent on the conversion of putrescine into polyamines and was short-lived. Within 6h after intraperitoneal administration of 0.8 mmol/kg body wt., ornithine decarboxylase activity had returned to normal values. This return correlated with the rapid loss of the diamines from the liver, and the decrease in activity could be slightly prolonged by treatment with aminoguanidine, a diamine oxidase inhibitor. A decrease in ornithine decarboxylase activity by these diamines was accompanied by the accumulation in the liver of a nondiffusible inhibitor that decreased the activity of a purified ornithine decarboxylase preparation. The possibility that administration of non-physiological diamines that are not converted into polyamines might be useful for the inhibition of polyamine synthesis is discussed.     

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.     

Regulation of ornithine decarboxylase activity by putrescine and spermidine in rat liver

The marked enhancement of the activity of ornithine decarboxylase (EC 4.1.1.17) in rat liver at 4 h following partial hepatectomy or the treatment with growth hormone could be almost completely prevented by intraperitoneal administration of putrescine. A single injection of putrescine to partially hepatectomized rats caused a remarkably rapid decline in the activity of liver ornithine decarboxylase with an apparent half-life of only 30 min, which is almost as rapid as the decay of the enzyme activity after the administration of inhibitors of protein synthesis. Under similar conditions putrescine did not have any inhibitory effect on the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) or tyrosine aminotransferase (EC 2.6.1.5). Spermidine given at the time of partial hepatectomy or 2 h later also markedly inhibited ornithine decarboxylase activity at 4 h after the operation and, in addition, also caused a slight inhibition of the activity of adenosylmethionine decarboxylase.     

Induction of spermidine/spermine N1-acetyltransferase in needle-punctured rat lens as a model of traumatic cataract

Isolated rat lens was punctured with a needle at a single point in the equatorial region and was incubated at 37 degrees C. Spermidine/spermine N1-acetyltransferase activity was increased about 5-fold at 8 h after the puncture. Concomitantly, putrescine content in the lens increased markedly at 8-16 h after the puncture, while spermidine levels were slightly depressed. Pretreatment of the lens with actinomycin D or cycloheximide blocked the increases of spermidine/spermine N1-acetyltransferase activity and putrescine content. Ornithine decarboxylase, on the other hand, was not induced to a detectable degree by this stimulus and 5 mM difluoromethylornithine could not block the increase of putrescine content. Polyamine oxidase showed a relatively constant activity that was sufficient for the metabolism of newly formed N1-acetylspermidine. These results suggested that, in the punctured lens, the polyamine levels were regulated predominantly by the activity of spermidine/spermine N1-acetyltransferase, but not by the induction of ornithine decarboxylase.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Conversion of exogenous spermidine into putrescine after administration to rats

Administration of large, but non-toxic doses of spermidine (0.4–1.25 mmol/kg) led to a substantial increase in putrescine in liver, kidney and a number of other tissues including muscle. The increase in putriscine peaked at 6 h after treatment and was completely prevented by administration of cycloheximide 3 h after the spermidine suggesting that the induction of a new protein was required. This protein is likely to be spermidine N¹-acetyltransferase which was induced by the treatment with spermidine and increased 3–4-fold in liver and kidney within 6 h. N¹-Acetylspermidine was detected in tissues at this time after spermidine treatment and experiments in which labeled spermidine was given indicated that a substantial fraction of the administered spermidine was converted into N¹-acetylspermidine and into putrescine. These results suggest that the rise in putrescine after spermidine treatment is brought about by the production of N¹-acetylspermidine which is converted into putrescine by the action of polyamine oxidase. The limiting step in this conversion is the activity of the acetylase which is induced in response to the rise in spermidine content. The acetylase/oxidase pathway, therefore, provides a means by which polyamine levels can be regulated and excess polyamine disposed of.     

Concentrations of putrescine and polyamines and their enzymic synthesis during androgen-induced prostatic growth

1. Castration of adult rats resulted in marked decreases in the amounts of putrescine, spermidine and spermine in the ventral prostate gland. Spermidine concentrations decline rapidly over the first 11 days after androgen withdrawal, reaching a value of only 12% of normal controls. Spermine concentrations diminish more slowly, reaching 24% of normal within 11 days. The spermidine/spermine molar ratio falls from 0.9 to 0.46 under these conditions. Putrescine concentrations decrease by 70% at 7 days after castration and then remain constant for some days. 2. After daily injections of testosterone propionate to rats castrated 7 days previously, prostatic spermidine and putrescine concentrations increase significantly within 24h; normal or even greater values are observed within 8 and 4 days respectively. In contrast, the spermine concentration does not increase until 5 days after commencement of androgen treatment. 3. The activities of two enzymes involved in polyamine biosynthesis (ornithine decarboxylase and a putrescine-activated S-adenosyl-l-methionine decarboxylase system) were greatly decreased soon after castration: after 7 days the respective values were 15% of normal for ornithine decarboxylase and 7% of normal for putrescine-dependent decarboxylation of S-adenosyl-l-methionine. Injection of testosterone propionate into animals castrated 7 days previously induced a rapid increase in both enzymic activities: ornithine decarboxylase was doubled in 6h, and increased three- to four-fold within 48h, whereas the putrescine-dependent decarboxylation of S-adenosyl-l-methionine doubled in 3h and increased tenfold within 48h of commencement of daily androgen treatments. 4. The activity of these enzyme systems was very low in the ventral prostates of hypophysectomized rats and was increased by administration of testosterone in a manner similar to that found in castrated rats. 5. Alterations in the activity of two ventral-prostate enzymes involved in ornithine production (arginase) and utilization (ornithine–2-oxoglutarate transaminase) that result from changes in the androgenic status of rats are described. 6. The findings presented suggest that the activities of ornithine decarboxylase and the putrescine-dependent S-adenosyl-l-methionine decarboxylase system, rather than ornithine concentrations, are rate-limiting for the formation of putrescine and polyamines in rat ventral prostate. 7. The relation of polyamines to androgen-induced prostatic growth is discussed with particular reference to the biosynthesis of proteins and nucleic acids.     

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.     

Temporal expression of ornithine decarboxylase in developing embryos of Musca domestica

The objective of this study was to partially characterize and follow the temporal expression of the enzyme ornithine decarboxylase (ODC) throughout embryonic and early larval development of Musca domestica. Enzymatically active ODC was shown to be present at detectable levels in the embryos only during the latter stages of embryogenesis. This temporally expressed enzyme displayed maximum activity at the time of hatching, and the activity rapidly declined in the newly hatched larvae. The half-life of ODC activity in extracts at the time of hatching and 30 min after hatching was 57 min and 12 min, respectively. The subunit molecular weight of the embryonic ODC was determined to be 46,000, and the apparent native molecular weight was determined to be 276,000. The concentrations of the polyamines putrescine, spermidine, and spermine also were determined throughout embryogenesis. Spermidine was found to be present in the embryos at about 10-fold higher concentrations than spermine and at about 100-fold higher concentrations than putrescine. These polyamines did not undergo major changes in concentration throughout development of the embryos.     

Polyamine and amino acid content, and activity of polyamine-synthesizing decarboxylases, in liver of streptozotocin-induced diabetic and insulin-treated diabetic rats

1. Concentrations of polyamines, amino acids, glycogen, nucleic acids and protein, and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, were measured in livers from control, streptozotocin-diabetic and insulin-treated diabetic rats. 2. Total DNA per liver and protein per mg of DNA were unaffected by diabetes, whereas RNA per mg of DNA and glycogen per g of liver were decreased. Insulin treatment of diabetic rats induced both hypertrophy and hyperplasia, as indicated by an increase in all four of these constituents to or above control values. 3. Spermidine content was increased in the livers of diabetic rats, despite the decrease in RNA, but it was further increased by insulin treatment. Spermine content was decreased by diabetes, but was unchanged by insulin treatment. Thus the ratio spermidine/spermine in the adult diabetic rat was more typical of that seen in younger rats, whereas insulin treatment resulted in a ratio similar to that seen in rapidly growing tissues. 4. Ornithine decarboxylase activity was variable in the diabetic rat, showing a positive correlation with endogenous ornithine concentrations. This correlation was not seen in control or insulin-treated rats. Insulin caused a significant increase in ornithine decarboxylase activity relative to control or diabetic rats. 5. S-Adenosylmethionine decarboxylase activity was increased approx. 2-fold by diabetes and was not further affected by insulin. 6. Hepatic concentrations of the glucogenic amino acids, alanine, glutamine and glycine were decreased by diabetes. Their concentrations and that of glutamate were increased by injection of insulin. Concentrations of ornithine, proline, leucine, isoleucine and valine were increased in livers of diabetic rats and were decreased by insulin. Diabetes caused a decrease in hepatic concentration of serine, threonine, lysine and histidine. Insulin had no effect on serine, lysine and histidine, but caused a further fall in the concentration of threonine.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.     

Response of enzymes involved in the metabolism of polyamines to phytohaemagglutinin-induced activation of human lymphocytes.

The stimulation of lymphocyte ornithine decarboxylase and adenosylmethionine decarboxylase produced by phytohaemagglutinin was accompanied by an equally marked, but delayed, stimulation of spermidine synthase, which is not commonly considered as an inducible enzyme. In contrast with the marked stimulation of these biosynthetic enzymes, less marked changes were observed in the biodegradative enzymes of polyamines in response to phytohaemagglutinin. Diamine oxidase activity was undetectable during all stages of the transformation. The activity of polyamine oxidase remained either constant or was slightly decreased several days after addition of the mitogen. The activity of polyamine acetylase (employing all the natural polyamines as substrates) distinctly increased both in the cytosolic and crude nuclear preparations of the cells during later stages of mitogen activation. Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, although powerfully inhibiting ornithine decarboxylase, produced a gradual enhancement of adenosylmethionine decarboxylase activity during lymphocyte activation, without influencing the activities of the two propylamine transferases (spermidine synthase and spermine synthase).     

Effect of inhibition of polyamine synthesis on the content of decarboxylated S-adenosylmethionine

1. The content of decarboxylated S-adenosylmethionine (AdoMet) in transformed mouse fibroblasts (SV-3T3 cells) was increased 500-fold to about 0.4fmol/cell when ornithine decarboxylase was inhibited by α-difluoromethylornithine. This increase was due to the absence of putrescine and spermidine, which serve as substrates for aminopropyltransferases with decarboxylated AdoMet as an aminopropyl donor, and to the enhanced activity of AdoMet decarboxylase brought about by depletion of spermidine. The increase in decarboxylated AdoMet content was abolished by addition of putrescine, but not by 1,3-diaminopropane. 2. 5′-Methylthiotubercidin also increased decarboxylated AdoMet content, presumably by direct inhibition of aminopropyl-transferase activities, but the increase in its content and the decline in spermidine content were much less than those produced by α-difluoromethylornithine. 3. Decarboxylated AdoMet content of regenerating rat liver was measured in rats treated with inhibitors of ornithine decarboxylase. The content was increased by 60% 32h after partial hepatectomy in control rats, by 90% when α-difluoromethylornithine was given to the partially hepatectomized rats, and by 330% when 1,3-diaminopropane was used to inhibit putrescine and spermidine synthesis. After 48h of exposure to 1,3-diaminopropane, which completely prevented the increase in spermidine after partial hepatectomy, there was a 5-fold rise in hepatic decarboxylated AdoMet concentration. These increases were prevented by treatment with putrescine or with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase. 4. These results show that changes in AdoMet metabolism result from the administration of specific inhibitors of polyamine synthesis. The possible consequences of the accumulation of decarboxylated AdoMet, which could, for example, interfere with normal cellular methylation or lead to depletion of cellular adenine nucleotides, should be considered in the interpretation of results obtained with such inhibitors.     

Additive stimulation of hepatic putrescine production by glucagon and insulin after partial hepatectomy in rats

When rats received glucagon or insulin every 2 h after partial hepatectomy (Hx), hepatic putrescine content was increased above control levels at 6 and 12 h, respectively. When the two hormones were combined, the increased levels were additive. Hepatic ornithine decarboxylase activity was above control levels at 12 h after insulin treatment. Hepatic spermidine N1-acetyltransferase activity was enhanced at 6 h only when glucagon was dosed. Putrescine administration from 0 to 4 h or from 6 to 10 h increased hepatic DNA synthesis to similar levels 22 h after Hx. These results suggest that glucagon and insulin additively stimulate hepatic putrescine production after Hx. This may explain the cooperative stimulation of liver regeneration by both hormones.