μM Spermidine Research Articles

Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.

Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo.

Structure of viral φ29 DNA condensed by simple triamines: A light‐scattering and electron‐microscopy study

AbstractThe structures of viral φ29 DNA condensed by triamines, principally spermidine, in 10−3M NaCl were investigated by static and dynamic light scattering and electron microscopy. All of the results for DNA condensed in 30 μM spermidine at neutral pH are quantitatively consistent with a toroidal structure with a mean outer diameter of 1850 Å. At pH 10.2, however, condensed structures of a completely different size and shape are observed for the first time. These structures are also more irregular in shape and more polydisperse than those at neutral pH. This conformational change is believed to result from a change in the mode of spermidine binding that is coupled to, or associated with, the (premature) titration of protons on the base‐ring nitrogens of guanine and thymine. Besides spermidine, certain homologs of spermidine, in which the butyl moiety of spermidine was replaced by longer pentyl through octyl moieties, were also studied. Though all of the triamines condensed the DNA at 30 μM, aggregation became a more prevalent occurrence as the length of the end chain increased. This suggests that crosslinking may play an important role in the condensation process. Finally, these aggregates are dissociated to a considerable extent at pH 10.2, and the resulting compact structures appear to be quite similar, independent of the triamine used to condense the DNA. The observed partial breakdown of aggregates is also consistent with the hypothesis of a change in mode of triamine binding at pH 10.2.

Initial stages of endogenous DNA degradation in rat liver nuclei isolated and incubated in the presence of Mg 2+ or polyamines

1. 1. This paper compares electrophoretic patterns of initial endogenous DNA degradation products, extracted from rat liver nuclei isolated and incubated in the presence of 5 mM of MgCl 2 or polyamines—0.15 mM of spermine and 0.5 mM of spermidine—for selected periods of time at 37° C. 2. 2. It was found that polyamines significantly slowed down the rate of DNA degradation in rat liver nuclei and did not essentially affect the pattern of supranucleosomal step of DNA breakdown. 3. 3. The differential stability of DNA in magnesium and polyamine nuclei were better expressed in the course of long-term incubation of nuclei at 4°C.

Activation of mammalian DNA ligase by polyamines

The activity of calf thymus DNA ligase was stimulated 2- to 3-fold by the addition of putrescine, spermidine and spermine. The optimal concentrations were 5 mM for putrescine, 0.5 mM for spermidine and 0.05–0.1 mM for spermine. Spermidine not only lowered the apparent Km value for Mg 2+ but also elevated the V value, whereas the apparent Km value for ATP was not changed. In the presence of Mn 2+ as a divalent cation, the V value was stimulated 3-fold by 0.5 mM spermidine. Monovalent cations, such as KCl and NaCl could replace the polyamines at the optimal concentrations of 50 mM and diminish the stimulation by the polyamines.

Enhancing effect of magnesium ion on cell-free synthesis of read-through protein of bacteriophage Qβ

This report describes the enhancing effect of magnesium ion on the synthesis of read-through protein of bacteriophage Qβ in a cell-free protein synthesizing system from E. coli . At 6 mM of magnesium acetate, the major product was coat protein. At 12 mM of magnesium, it was replaced by read-through protein. This enhanced synthesis was substituted by the addition of 0.25 mM of spermine or 1 mM of spermidine to 6 mM of magnesium. These results suggest that magnesium or combination of magnesium and polyamines causes leaky termination at the end of the coat protein cistron of Qβ-RNA.

Influence of polyamines on the activity of DNA polymerase I from Escherichia coli

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A—T) n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl 2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA) n · (dT) 10 and (rA) n · (dT) 10 (20 : 1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA) n · (dT) 10 and somewhat less for (dA) n · (dT) 10. The apparent K m for the primer (rA) 10 · (dT) 10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent K m for dTTP. The influence of polyamines on both the 5′ → 3′ and 3′ → 5′ nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A—T) n and T 7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.

Inhibition of phosphorylase phosphatase by polyamines

Phosphorylase phosphatase from rabbit liver or from bovine heart, purified as the M r 35,000 form, is inhibited by the naturally occurring polyamines, spermine and spermidine. The inhibition is apparently competitive with a K i of 0.037 mM for spermine and 0.65 mM for spermidine. Further investigation showed that the inhibition is substrate-directed, i.e. due to an interaction of the polyamines with the rabbit muscle phosphorylase a used as the substrate.

Purification and characterization of two tRNA-(guanine)-methyltransferases from rat liver

tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA( N 2-guanine)-methyltransferase I (EC 2.1.1.32) were isolated from rat liver. The (guanine-1)-methyltransferase preparation is 6800-fold purified and is free from contaminating methyltransferases or ribonuclease. The molecular weight of (guanine-1-)-methyltransferase is 83 000. Of seven purified Escherichia coli tRNAs examined, only tRNA Met f was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNA Met f is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the K m for S-adenosylmethionine is 3 μM and K i for S-adenosylhomocysteine is 0.11 μM for (guanine-1-)-methyltransferase. ( N 2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNA Phe, tRNA Val and tRNA Arg are substrates for this enzyme which introduces a methyl at the 2-amino group of the guanine at position 10 from the 5′-terminus of these tRNAs. The methylation of tRNA Phe is maximally stimulated by 100 μM spermidine with a pH optimum of 8.0. ( N 2-Guanine-)-methyltransferase has a K m for S-adenosylmethionine of 2 μM and a K i for S-adenosylhomocysteine of 23 μM with E. coli K-12 tRNA as methyl acceptor.

Effects of polyamines on in vitro dna synthesis by DNA polymerases from calf thymus.

DNA synthesizing reactions catalyzed both large and small species of calf thymus DNA polymerase (DNA polymerase-alpha and -beta) [EC 2.7.7.7] were stimulated to comparable extents by the presence of spermidine or spermine, and it was not possible to differentiate these two species in terms of their sensitivities to polyamines. Optimal concentrations for stimulation were 0.5-1.0 mM for spermidine and 2-10 mjM for spermine. Excess polyamines strongly inhibited the reactions. The modes of stimulation were as follows: 1) Stimulation was observed with templates bearing long single-stranded sections when either natural DNA or synthetic homopolymer-oligomer duplex was used. 2) The nautral DNA-dependent reaction was stimulated by polyamines at suboptimal concentrations of Mg2+; the apparent Km value for Mg2+ was lowered on adding polyamines, while the Vmax value was unchanged. When synthetic homopolymer-oligomer duplex was used as a template, the reaction was stimulated spermidine even at the optimal concentration of Mn2+. 3) Polyamines markedly influenced the salt requirements of the reactions of DNA polymerase. Spermidine could replace salts such as KC1 or NaC1 at concentrations less than 1/100 of those of salts.

DNA-dependent RNA polymerases I and II from kidney. Effect of polyamines on the in vitro transcription of DNA and chromatin.

DNA-dependent RNA polymerases I and II were purified from pig kidney nuclei by chromatography on DEAE-Sephadex and phosphocellulose. When nonlimiting amounts of double-stranded DNA were used as the template, the in vitro transcription was markedly stimulated by spermidine and spermine. Maximal stimulation of RNA polymerase I occurred at 2-5 mM spermidine and 0.5-2 mM spermine, whereas optimal polyamine concentrations for RNA polymerase II were 5-10 and 1-5 mM for spermidine and spermine, respectively. DNA transcription by polymerase II was stimulated to a greater extent than that of polymerase I. Higher spermine (5-10 mM) concentrations were strong inhibitors of both polymerases under these conditions. The apparent Km of RNA polymerases I and II for UTP was unchanged at optimal polyamine concentration; under the same conditions the maximal reaction velocity was increased two- to three-fold and was essentially due to an increase in the rate of chain elongation. Thus, in a typical experiment the average chain length as determined by the UMP/uridine ratio increased from 570 to 1330 and the chain elongation rate increased from 0.64 to 1.44 nucleotides times sec-1 in the presence of spermine. When limiting quantities of native DNA were employed as the template, both RNA polymerases I and II were inhibited by 1-2 mM spermine. Kidney chromatin could be transcribed by homologous RNA polymerases with an efficiency ranging from 2 to 10% of that with native DNA. When chromatin was used in nonlimiting amounts instead of DNA, RNA polymerase II activity was again stimulated about two-fold at 2 mM spermine. Under these conditions, RNA polymerase I activity was inhibited by spermine. The inhibition of RNA synthesis in vitro at limiting quantities of templates (DNA or chromatin) could be overcome by preincubation of the enzyme with templates before polyamines were added. This inhibition thus appears to be due to a block in the initiation of RNA chains. Similar inhibition of transcription by RNA polymerase II was also observed with limiting quantities of chromatin as the template.

Polyamines and polyamine-metabolizing enzyme activities in human semen

The content of polyamines putrescine, spermidine and spermine as well as the activities of some polyamine-metabolizing enzymes have been analyzed from normal and pathological human semen samples. Seminal fluid from semen samples showing no apparent abnormalities in semen analyses contained about 0.2 mM of putrescine, 0.1 mM of spermidine and 3 mM of spermine. Seminal plasma was found to be an exceptionally rich source of diamine oxidase activity (EC 1.4.3.6). In addition, polyamine-synthesizing enzyme activities, S-adenosylmethionine decarboxylase and spermidine synthase, were invariably found in seminal plasma. Spermatozoa contained very low or undetectable activities of 5-adenosylmethionine decarboxylase and spermidine synthase; however, a definitive diamine oxidase activity was found also in the cellular component of the semen. The activity of diamine oxidase, and especially that of spermidine synthase, was relatively high in semen samples having low sperm density (less than 20 × 10 6 spermatozoa per ml). With increasing number of spermatozoa there was a slight decrease in both activities, but as the sperm count exceeded 100 × 10 6 per ml the activity of diamine oxidase and spermidine synthase sharply increased. The concentration of spermine in seminal plasma showed only minor changes in relation to the number of spermatozoa in semen, the changes being roughly opposite as compared to the changes of the enzyme activities. A significant negative correlation was found between the concentration of putrescine and the activity of diamine oxidase in seminal plasma. Several properties of diamine oxidase from human seminal plasma were found to be comparable to those of other mammalian diamine oxidases. Using radioactive putrescine as the substrate the enzyme activity was inhibited by addition of unlabelled histamine, cadaverine, spermidine and spermine. The enzyme was also powerfully inhibited by various carbonyl reagents and methylglyoxal bis-(guanylhydrazone).