Purification of spermine synthase from bovine brain by spermine-Sepharose affinity chromatography.
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Effects of spermine on the detection of induced forward mutation at the Can1 locus in yeast: evidence for selection against canavanine-resistant mutants.
The effect of exogenous spermine tetrahydrochloride (0.5 mg/ml) on hydrazine- and nitrous acid-induced forward mutation to canavanine resistance (CAN1 leads to can1, normal to defective arginine permease) was examined in stationary-phase haploid Saccharomyces cerevisiae. Post-treatment cell division (specifically DNA replication) is required for hydrazine mutagenesis at this locus, whereas nitrous acid mutagenesis exhibits, in addition, a significant post-treatment-independent component. Spermine addition only during mutagenic treatments in buffer did not affect mutagen cytotoxicity, but did result in a slight yet consistent decrease in induced mutation frequencies. Addition of spermine to the yeast extract--peptone--dextrose (YEPD) post-treatment growth medium resulted in dramatic reductions of induced mutation frequencies, which could be alleviated by pregrowth in spermine-containing YEPD. Such a medium was found to cause an apparent temporary growth inhibition for almost 40 h, after which the growth rate of the culture increased rapidly. Cultures "recovering" from spermine inhibition were no longer inhibitable by spermine in fresh medium, suggesting an outgrowth of spontaneous and/or induced spermine-resistant derivatives. Genetic analysis of one isolate revealed a single dominant nuclear gene conferring resistance by some means other than defective spermine uptake. Growth of this mutant was only slightly inhibited by spermine (20% increase in doubling time), while mutation expression remained high. Results of competitive growth experiments indicated that spermine-containing YEPD exerted a selection pressure against canavanine-resistant cells, while YEPD by itself did not. The mechanism for this selection is not presently understood. With respect to replication-dependent induced mutation at CAN1, our initial observation of a strong apparent antimutagenic action of spermine was found to be best explained by this specific selection against can1 mutants. This underscores the need for caution in the interpretation of experiments designed to study physiological modification of mutagenic potential.
Canavanine↗
Specific effects of spermine on ouabain-sensitive and potassium-dependent phosphatase activity of kidney plasma membranes. Specificity of the potassium sites.
Specific inhibition of ouabain-sensitive and K+-dependent p-nitrophenyl-phosphatase activity of rabbit kidney plasma membranes by spermine (N,N'-bis(3-aminopropyl)-1,4-butanediamine) was characterized kinetically. 1. Inhibition by spermine was competitive with K+. The Ki for spermine was 31 micronM in the presence of 1 mM Mg2+. 2. Excess Mg2+ inhibited the ouabain-sensitive phosphatase activity in competition with K+. The Ki for Mg2+ was 2.6 mM. 3. Increasing Mg2+ concentrations reduced the spermine inhibition. This could be observed at Mg2+ concentrations higher than that of K+. 4. In the absence of inhibition by Mg2+, spermine was noncompetitive with Mg2+ which was essential for the ouabain-sensitive phosphatase activity. This could be observed at Mg2+ concentrations lower than that of K+. 5. Although Ca2+ was a strong inhibitor of the ouabain-sensitive phosphatase activity in the presence of K+, it produced a small stimulation of the activity in the absence of K+. Approximately 0.1 mM Ca2+ gave the maximum stimulation. 6. The observed Ca2+- and Mg2+-dependent phosphatase activity was inhibited strongly by ouabain and by spermine. The half-maximal inhibition concentrations of ouabain and spermine were 0.1 and 63 micronM, respectively. It is likely that Mg2+, Ca2+ and spermine bind to the same site as does K+.
4-Nitrophenylphosphatase↗
The role of polyamines in the aminoacyl transfer ribonucleic acid synthetase reactions. Demonstration of the requirement for magnesium ion and a secondary stimulatory effect of spermine.
Spermine and related polyamines have been reported to substitute for Mg2+ in the aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases, but not in the ATP-PP-i exchange reaction. Such observations have led some workers to propose that these reactions proceed via a concerted mechanism rather than the usual two-step mechanism involving an aminoacyladenylate intermediate. In an attempt to elucidate the mechanism of the spermine effect on acylation and exchange, both reactions were re-examined using isoleucyl-tRNA synthetase from Escherichia coli. In the absence of added Mg2+ untreated tRNA was acylated in the presence of spermine, but tRNA from which Mg2+ had been scrupulously removed was not. ATP-PP-i exchange was not observed when spermine was used in place of Mg2+; however, if tRNA possessing sequestered Mg2+ was added, the exchange reaction was observed. These data suggest that a primary effect of spermine is to displace bound Mg2+ from tRNA in quantities sufficient to promote both the ATP-PP-i exchange and esterification of tRNA. The previously reported stimulatory effects of polyamines on these reactions are believed to be artifacts due to Mg2+ contamination of tRNA. Providing trace levels of Mg2+ are present, spermine exerts a secondary stimulation of the rate of aminoacylation, the mechanism of which is unknown. The results presented refute arguments that these enzymes proceed by a concerted mechansim and support the intermediacy of aminoacyladenylates.
Adenosine Triphosphate↗
Effects of spermine on activity and stability of calcium-dependent guanosine 3',5'-monophosphate phosphodiesterase.
Spermine in micromolar concentrations decreased the basal activity of a guanosine 3',5'-monophosphate (cGMP) phosphodiesterase from bovine brain but had no effect in the presence of Ca2+ plus the calcium-dependent regulatory protein (CDR) which increased the activity of the enzyme 4- to 6-fold. Similar effects of spermine were observed on the enzyme at several stages of purification. Spermidine and putrescine were also inhibitory but higher concentrations were required. In the absence of Ca2+ and CDR, the enzyme exhibited two apparent Km values for cGMP (2.5 and 20 microM) which were unaltered by spermine. In the presence of Ca2+ and CDR (when spermine had no effect on activity), a single Km (3.5 microM) was observed. Enzyme purified by chromatography on CDR-Sepharose was rapidly inactivated during incubation at 30 degrees C in 5 mM potassium phosphate buffer (pH 7.0) with EDTA and ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Spermine (20 microM) partially stabilized enzyme activity under these conditions, although it was somewhat less effective than 2 mM MgCl2. The inhibitory effects of spermine (or other polyamines) on basal phosphodiesterase activity, which can be overcome by Ca2+ and CDR, could be important in the regulation of cellular cyclic nucleotide content.
3',5'-Cyclic-GMP Phosphodiesterases↗
Selective polyamine-binding proteins. Spermine binding by an androgen-sensitive phosphoprotein.
Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S.
Androgens↗
The effects of spermine and Mg2+ on the catalytic mechanism of isoleucine: tRNA ligase.
Isoleucyl-tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH-range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects. 2. The spermine-stimulated reaction has a steeper temperature-dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl-tRNA formation are detected in the presence of Mg2+ plus or minus spermine, but only a single step is observed in the presence of spermine alone. Thus the rate-limiting steps under normal assay conditions are different for the two cations. 3. Enzyme-bound isoleucyl-AMP can be formed in the absence of Mg-2+ and plus or minus spermine. 4. It is concluded that there is no evidence for cation-dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.
Enzyme Activation↗
Mechanism of spermine stimulation of rat-liver isoleucyl-tRNA formation.
The mechanism of spermine stimulation of rat-liver isoleucyl-tRNA formation has been studied by Cleland's method [Biochim. Biophys. Acta, 67, 104--137 & 173--197 (1963)]. It was demonstrated that isoleucyl-tRNA formation proceeded through a ping-pong mechanism in the presence of spermine and magnesium. From a study of the effect of spermine on both the formation of isoleucyl-AMP . enzyme complex and on the subsequent formation of isoleucyl-tRNA from the isoleucyl-AMP . enzyme complex, evidence is presented that the stimulatory effect of spermine functions mainly through the spermine preventing the tRNA inhibition of isoleucyl-AMP . enzyme complex formation.
Adenosine Triphosphate↗
Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain.
Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocystemine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88 000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km values for S-methyladenosylhomocysteamine was 0.6 microM and about 60 microM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor. Both the reaction products, spermine and 5'-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosylhomocysteamine (Ki value of about 0.3 microM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 mM). Spermine synthase had no requirements for metal or other cofactors.
Animals↗
The synthesis and turnover of spermidine and spermine in mouse brain.
Following the administration to mice of radiolabeled putrescine by intraventricular injection, changes in the specific radioactivity of putrescine, spermidine, and spermine have been measured. Putrescine decline was biphasic, being more rapid over the first 12 hr(t 1/2 = 5 hr) than over the remainder of the 48-hr period (t 1/2 = 11 hr) that significant labeling was detected. Spermidine was rapidly labeled during the decline in putrescine radioactivity and maximum incorporation of label occurred at 18 hr. Subsequently, spermidine specific activity declined with a half-life of 22 days. Spermine synthesis was slower, with maximum labeling occurring after 4 days. Spermine turnover, measured at a time when spermidine radioactivity had substantially declined, was extremely slow (t 1/2 = 92 days). The data supports the view that putrescine is a precursor of spermidine which in turn is required for spermine synthesis.
Animals↗
Axonal transport of putrescine, spermidine and spermine in normal and regenerating goldfish optic nerves.
Radopactove putrescine, spermidine or spermine was injected into the right eye of normal goldfish and fish in which both optic nerves had been crushed 18 days earlier. Fish were sacrificed 0.25-21 days after injection. Trichloroacetic acid-soluble and -insoluble material was extracted from the right retina and both tecta and assayed for radioactivity (significant differences between left and right tecta suggesting axonal transport). The nature of the radioactivity in the TCA-soluble fraction was determined on an amino acid analyzer. Results indicate that putrescine is not axonally transported in intact goldfish optic nerves, but that during regeneration of the optic nerve large amounts of putrescine are axonally transported at rates similar to the fast component of protein transport. Spermidine appears to be axonally transported both in intact optic nerves and in regenerating optic nerves, and at an intermediate rate of transport; the amount of spermidine transported is significantly increased during regeneration. Spermine is also axonally transported in intact and regenerating nerves, at a rate similar to the rapid rate of protein transport. The amount of spermine transported appears to be slightly less in regenerating than in intact nerves during early stages of regeneration, but increases during later stages of nerve regeneration. The results suggest that putrescine and spermidine may be preferentially transported during nerve regeneration, while spermine and spermidine are transported extensively in intact nerves.
Animals↗
Plasma spermine concentrations of patients with benign and malignant tumours of the breast or prostate.
A radioimmunoassay has been developed for the measurement of plasma spermine concentrations. The sensitivity of the method is 1 pmol spermine/100 microliters plasma and the crossreactivity was 12% with spermidine and 0.18% with putrescine. Plasma spermine levels of patients with benign and malignant tumours of the prostate or breast were measured using this technique. Concentrations were only occasionally elevated in patients with prostatic tumours compared to normal individuals and there was no difference between those men with benign (mean concn. 0.21 +/- 0.14 nmol/ml plasma) or malignant (mean concn. 0.21 +/- 0.11 nmol/ml plasma) tumours. Only 17% of the patients with breast carcinoma had elevated levels of spermine, although there was a significant difference in the concentrations of the breast cancer group of patients compared to normals. No correlation was found between elevated plasma spermine concentrations and tumour grade or presence or spread of metastases in those patients.
Adolescent↗
Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation.
Serum polyamine oxidase (EC 1.4.3.4) is known to react in vitro with radio-labelled spermine4+ to produce di-oxidized spermine which must incorporate the label. Di-oxidized spermine was compatible with a radio-labelled compound2+ separated from the reaction mixture by ion-exchange chromatography. The compound was measured and had a half-life of about 2.3 h in tissue culture medium. It also rapidly and tightly bound to an unidentified serum component (gel-filtration chromatography indicated a complex of mol. wt 70,000) so that dissociation required treatment with strong acid (10N HCl). Findings suggest that the di-oxidized spermine, in either its free cationic or bound form, potently arrested cell proliferation. This arrest was non-cytotoxic and was confined to the G1 phase of the cell cycle. Products of di-oxidized spermine autodegradation, including trace amounts of stable and cytotoxic acrolein (arrested S phase), were unlikely to have contributed significantly to the arrest.
Acrolein↗
Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect.
The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40--60% within 20--40 h after castration. This effect is reversed very rapidly within 15--30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate.
Animals↗
Isolation and characterization of Saccharomyces cerevisiae mutants deficient in S-adenosylmethionine decarboxylase, spermidine, and spermine.
Four mutants were isolated from Saccharomyces cerevisiae that are deficient in S-adenosylmethionine decarboxylase (spe2). All four mutants are chromosomal and fall into a single complementation group tightly linked to arg1. Since one of the mutants contained a temperature-sensitive activity, this complementation group defines the structural gene. Mutants totally lacking enzymic activity did not contain spermidine or spermine and had a greatly increased doubling time when grown in the absence of these two polyamines. Addition of 10(-6) M spermidine or 10(-5) M spermine, but not putrescine or cadaverine, restored the doubling time to that of the wild type. Diploids formed from a cross of two mutants completely deficient in spermidine and spermine were unable to sporulate in the absence of added spermidine or spermine. We obtained evidence that arg1 was not located on any of the 17 known chromosomes, and therefore we postulate that arg1 and spe2 are located on a new 18th chromosome.
Adenosylmethionine Decarboxylase↗
Spermine hinders post-conjugational recombination in Escherichia coli K-12.
Treatment of Hfr and F- mixture with spermine during the first minutes of mating decreased the number of viable recombinants and inhibited functional recombinant structure formation in the intragenic cross lacz157Xlaczs. The treatment applied after 15 minutes of mating was ineffective. The decrease of recombination frequency was not caused by any interference in mating union formation. The efficiency of donor DNA transfer in the presence of spermine was increased two-fold. Spermine blocked the formation of S1-nuclease degradable DNA in the post-conjugant recipient cells. It is suggested that spermine probably interferes with the early steps of recombination, most likely by the stabilization of DNA against strand separation.
Conjugation, Genetic↗
Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine.
The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (ki value of 0.3 mM) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylase a.
Animals↗
Structural analysis of spermine and magnesium ion binding to yeast phenylalanine transfer RNA.
Refinement of the diffraction data at 2.5-A resolution from orthorhombic crystals of yeast tRNAPhe has proceeded to the point where spermine and magnesium ions can be located in the difference electron density map. Two spermine molecules are found: one is located in the major groove at one end of the anticodon stem; the other is near the variable loop and curls around phosphate 10 in a region where the polynucleotide chain takes a sharp turn. Four distinct magnesium ions have been identified: one in the anticodon loop, two in the D loop, and one coordinated with phosphates 8, 9, 11, and 12, where the polynucleotide chain is coiled. The conformation of the anticodon stem and loop is stabilized by the cations at the end of the molecule. The positions of these ions may be related to aspects of the biological activity of tRNA. The spermine and magnesium ions appear to be important in maintaining the overall folding of the tRNA molecule.
Anticodon↗