Influence Of Spermine Research Articles

THE INFLUENCE OF SPERMINE AND CHLORHEXIDINE ON THE SURVIVAL, ELECTROKINETIC AND CYTOMORPHOLOGICAL CHARACTERISTICS OF HUMAN PROSTATE CANCER CELLS

Summary. Aim: to study the effects of spermine and the spermine oxidase inhibitor chlorhexidine, applied alone or in combination, on the viability, electrokinetic and structural-functional characteristics of human prostate cancer cells in vitro. Object and methods: Studies were conducted on cell cultures: differentiated androgen-dependent LNCaP cell line and low-differentiated androgen-independent DU-145 cell line. Cell survival was determined by the method of exclusion of the vital dye trypan blue by living cells. The electrokinetic parameters of the cells (ζ-potential and total surface charge density) were determined according to the Smoluchovsky and Quincke-Helmholtz equations. Morphological changes were assessed by light microscopy of fixed preparations of cells stained with hematoxylin and eosin. Results: The fact established by previous studies that spermine at a concentration of 1.5 mM exhibits a pronounced inhibitory effect on the growth of differentiated human prostate cancer cells of the LNCaP line, which consists in reducing their survival to 48.5 ± 1.5% compared to intact control, and when this factor is used in a concentration exceeding 5.0 mM – in their death. It was shown that poorly differentiated cells of the DU-145 line were significantly more resistant to its influence: their survival decreased to 56.0 ± 1.9% only under the influence of spermine at a concentration of 5.0 mM; a concentration of 10.0 mM caused complete cell death of both lines. The trend persisted in the case of chlorhexidine: under its influence at a concentration of 15.0 μM, the survival of cells of the hormone-dependent LNCaP line was 33.0 ± 1.5%, and the hormone-independent DU-145 cell line was 79.0 ± 2.5%. The combined use of spermine and chlorhexidine (0.6 mM and 3.2 µM, respectively) caused a strong cytotoxic effect in cells of the LNCaP line (survival 19.0 ± 1.2%), while cells of the DU-145 line maintained a fairly high rate (76.0 ± 2.1%) even with a significantly higher content of spermine (3.0 mM) and chlorhexidine (5.0 μM) in the culture medium. It was established that under the influence of chlorhexidine, the cells of both studied lines show an inversion of the sign of the surface electric charge, changing it from negative to positive under physiological conditions, just as it was shown in experiments with spermine on cells of the LNCaP line. Certain quantitative differences caused by the investigated compounds in the electrokinetic indicators of cells of hormone-dependent and hormone-independent lines were revealed. Cells of the DU-145 line under the influence of spermine, or simultaneously spermine and chlorhexidine, showed a typical apoptotic morphology: they rounded and separated from each other and the substrate, chromatin condensation, blurred contours of the surface membrane, nuclear fragmentation and the presence of apoptotic bodies were noted. Conclusions: Spermine in concentrations of 1.5-10.0 mM and chlorhexidine in concentrations of 10.0-30.0 μM, applied separately, have a cytotoxic effect on human prostate cancer cells of LNCaP and DU-145 lines in culture. Cytotoxic properties of spermine, chlorhexidine or their combination are significantly more pronounced when acting on the hormone-dependent LNCaP line cells than on the hormone-independent DU-145 line. Their combined use demonstrates a significantly higher cytotoxic effect on cells of the LNCaP line, which is not observed in the DU-145 cell line. Chlorhexidine, like spermine, causes an inversion of the surface charge in the studied cell lines, changing its sign from negative to positive. The revealed differences like changes in survival and electrokinetic parameters of LNCaP and DU-145 cells can be explained by the presence or absence of androgen binding receptors on their surface membrane. Cells of the DU-145 line under the influence of spermine, or at the same time spermine and chlorhexidine, go to the state of apoptosis, as well as cells of the LNCaP line when they are exposed to spermine.

Influence of spermine and nitrogen deficiency on growth and secondary metabolites accumulation in Castilleja tenuiflora Benth. cultured in a RITA® temporary immersion system.

The effect of exogenous spermine (SPM) on Castilleja tenuiflora shoots developing under nitrogen deficiency (ND) stress was evaluated. Shoots cultivated in a temporary immersion system were subjected to four experimental treatments: (1) control; (2) exogenous SPM; (3) ND; and (4) ND+SPM. Shoots were longer in the ND+SPM treatment (6.3 ± 0.5 cm) than in the ND treatment (4.2 ± 0.5 cm). The total chlorophyll content was similar in the control and SPM treatments (0.41 µg mg−1 FM) and the highest values of total phenolic content were detected at 21 days in the ND+SPM treatment (84.1 ± 0.05 GAE g−1 DM). In the ND+SPM treatment, the phenylalanine ammonia lyase activity increased earlier than in ND treatment, and reached its maximum at day 21 (3.9 ± 0.2 µmol E‐CIN h−1 mg−1 protein). Compared with the control, the ND and ND+SPM treatments resulted in increased secondary metabolites contents in both root and aerial parts. The strongest effect was in the roots, where the SPM and ND+SPM treatments both resulted in increased quercetin content (4.3‐fold that in the control). Our results showed that SPM partially counteract the damage caused by ND and results in increased contents of valuable bioactive compounds.

Spermine as a porcine pancreatic elastase activator: spectroscopic and molecular simulation studies

The aim of this study was to investigate the spermine effect on the thermal denaturation, conformation and activity of elastase at three temperatures of 303, 313 and 323 K in the Tris buffer, at pH 8.5, using UV–vis spectrophotometry, spectrofluorometry and circular dichroism as well as molecular docking and molecular simulation. The increased absorption of elastase in the presence of spermine suggested a change in the environment of tryptophan. It was found that under the influence of spermine, the emission intensity of elastase extremely was reduced, and the use of the Stern-Volmer equation showed that some static quenching had occurred. The thermodynamic parameters values (enthalpy and entropy) and the molecular docking technique also revealed that van der Waals forces or hydrogen bonding interactions played an important role in the binding process. The spermine–elastase complex formation led to increasing the value of the catalytic constant (kcat). So it could be considered as an activator. Slight changes were observed in the second structure of elastase (1.06% increase for the α-helix and 0.048% decrease the β-sheet) and the thermal stability effect. Molecular docking results also demonstrated that spermine could bind to porcine pancreatic elastase, and van der Waals forces or hydrogen bonding interactions played the major role in the binding process. Overall, our results showed that spermine could induce structural alterations in elastase, acting as a partial stabilizer and an activator for the enzyme.Communicated by Ramaswamy H. Sarma

The influence of spermine on Ca2+-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,ω-hexadecanedioic acids

The effect of spermine on Ca(2+)-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,Ω-hexadecanedioic acid was studied. It has been shown that spermine inhibited the cyclosporin A-insensitive mitochondrial swelling induced by palmitic acid and Ca2+ and α,Ω-hexadecanedioic acid and Ca2+. 100 μM spermine did not influence the mitochondrial respiration in state V2 and the respiration stimulated by palmitic acid, α,Ω-hexadecanedioic acid and Ca2+. Pre-incubation of liposomes with 100 μM spermine resulted in the inhibition of palmitic acid/Ca(2+)- and α,Ω-hexadecanedioic acid/Ca(2+)-induced release of the fluorescent dye sulforhodamine B from liposomes. At the same time, spermine added to fatty acids-contained membranes of liposomes stimulated Ca(2+)-dependent release of sulforhodamine B from liposomes. It was shown that an addition of spermine to liposomes resulted in a significant increase in z-potential of liposomal membranes (from -39.8 mV to -18.6 mV). A possible mechanism of spermine influence on palmitic acid/Ca(2+)- and α,Ω-hexadecanedioic acid/Ca(2+)-induced permeability transition in mitochondria and liposomes is discussed.

Real‐Time Monitoring of New Delhi Metallo‐β‐Lactamase Activity in Living Bacterial Cells by 1H NMR Spectroscopy

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-β-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

Real‐Time Monitoring of New Delhi Metallo‐β‐Lactamase Activity in Living Bacterial Cells by 1H NMR Spectroscopy

AbstractDisconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo‐β‐lactamase subclass 1 (NDM‐1), an emerging antibiotic‐resistance threat. Cell‐based NMR studies demonstrated that two known NDM‐1 inhibitors, L‐captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM‐1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM‐1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole‐cell settings, including mammalian cells.

The influence of spermine and hydrogen peroxide on programmed cell death in tuberculate rhizoids ofMarchantia polymorpha

Evidence for naturally occurring programmed cell death in Marchantia polymorpha tuberculate rhizoids as a function of age is described. Membrane degradation and DNA fragmentation, two major events that occur during programmed cell death, are detected and quantified using Trypan Blue staining and the TUNEL Assay. Membrane permeability and DNA fragmentation both increase as the tuberculate rhizoids aged. Spermine suppressed this response; hydrogen peroxide accelerated it. This research has advanced the study of PCD by demonstrating that nutrient recycling of the thallus and tuberculate rhizoids of Marchantia polymorpha are interrelated.

The Influence of Spermine on the Activity of a Protein Kinase C-like Enzyme in Marchantia polymorpha Thalli During Programmed Cell Death

Abstract Protein Kinase C-like activity was established in Marchantia polymorpha. Different concentrations were detected in young and old tissue extracts from untreated and spermine-treated thalli after agarose gel electrophoresis in the presence of a PKC-specific fluorescent tags. Cytosolic putative PKC concentrations (nmol/g tissue) in untreated young thalli were higher than concentrations in old tissue. Old spermine-treated tissue possessed higher cytosolic putative PKC concentrations than young untreated thalli. Both young and old spermine-treated tissue extracts possessed higher total protein levels than young and old untreated tissue extracts. The data suggest that spermine-treatment evoked a qualitative and quantitative decrease in senescence.

Influence of Spermine on DNA Conformation in a Molecular Dynamics Trajectory of d(CGCGAATTCGCG)2: Major Groove Binding by One Spermine Molecule Delays the A→B Transition

The effect of spermine on the A-DNA to B-DNA transition in d(CGCGAATTCGCG)2 has been investigated by five A-start molecular dynamics simulations, using the Cornell et al. potential. In the absence of spermine an A→B transition is initiated immediately and the DNA becomes equidistant from the A- and B-forms at 200ps. In three DNA-spermine simulations, when a spermine is located across the major groove of A-DNA in one of three different initial locations, the time taken to reach equidistance from the A- and B-forms is delayed until 800, 950 or 1000ps. In each case the A-form appears to be temporarily stabilized by spermine's electrostatic interactions with phosphates on both sides of the major groove. The onset of the A→B transition can be correlated with the spermine losing contact with phosphates on one side of the groove and with A-like → B-like sugar pucker transitions in the vicinity of the spermine bridge. However in the fifth trajectory, in which the spermine initially threads from the major groove via the backbone into the minor groove, the B→A transition occurs rapidly once again and the DNA is equidistant between the A- and B-forms within 300ps. This indicates that the mere presence of spermine is insufficient to delay the transition and that major groove binding stabilizes A-DNA.

Influence of spermine on intestinal maturation of the glycoprotein glycosylation process in neonatal rats

Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an alpha-1, 2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in alpha-1,2-fucosyltransferase level and of the decrease in alpha-l-fucosidase level after treatment with spermine, several alpha-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development.

Influence of spermine on intestinal maturation of the glycoprotein glycosylation process in neonatal rats

Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an α-1,2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in α-1,2-fucosyltransferase level and of the decrease in α-L-fucosidase level after treatment with spermine, several α-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development.

Polyamine induced Z-conformation of native calf thymus DNA

Conformational isomerization of native calf thymus DNA under the influence of spermine, spermidine and putrescine was monitored by UV absorption and immunospecific anti-Z-DNA antibodies. Immunological data indicated increased binding of anti-Z-DNA antibodies to polyamine-perturbed conformations of native DNA and double stranded poly(dG-dC). In the absence of polyamines, anti-Z-DNA antibodies did not bind to either polymers. Analysis of UV absorption studies indicates a left handed conformation of nDNA in the presence of polyamines. Moreover, we observed total aggregation of DNA in the presence of spermine on prolongued incubation. These perturbations in conformation were dependent on polyamine concentration. The results clearly suggest that certain regions of nDNA are sensitive to elevated levels of polyamines and are capable of undergoing B → Z transition.

Dual effect of spermine on acrosomal exocytosis in capacitated bovine spermatozoa

The influence of spermine on the acrosomal exocytosis of capacitated bovine spermatozoa was studied. Dual effect of spermine was observed, depending on its concentration. It was shown that 10 μ,M spermine stimulated acrosomal exocytosis and prostaglandin F 2α production, whereas higher concentrations of spermine inhibited these processes. Acrosomal exocytosis induced by spermine was inhibited by staurosporine, a specific protein kinase C (PKC) inhibitor, indicating that PKC may be involved in this stimulation. Also, acrosomal exocytosis induced by the PKC activator phorbol 12-myristate- l3-acetate was inhibited by 10 mM spermine. Therefore, these data indicate that spermine is involved in signal transduction events leading to exocytosis. We suggest that the concentration-dependent reversal of the stimulatory action of spermine could be explained by the existence of two binding sites for spermine: high affinity sites involved in inducing acrosomal exocytosis by low spermine concentration and low affinity sites mediating inhibition of acrosomal exocytosis by high concentration of spermine.

The influence of exogenously supplied spermine on protochlorophyllide and chlorophyll biosynthesis

The pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus synthesizes only protochlorophyllide and traces of chlorophyll in darkness. The reduction of protochlorophyllide to chlorophyllide and the synthesis of chlorophyll take place in the light. The tetramine spermine, contrary to the diamine putrescine and the triamine spermidine, regulates the amount of protochlorophyllide and chlorophyll in the dark and in the light. Low amounts of exogenous spermine induce chlorophyll biosynthesis (the optimum level is 0.32 mM SPM), while higher amounts inhibit chlorophyll biosynthesis. In the dark, spermine enhances the amount of accumulated protochlorophyllide, a fact that we attribute to inhibition of the light-independent protochlorophyllide oxidoreductase activity. Uptake measurements of spermine and its possible incorporation in prolamellar bodies/prothylakoids in comparison with the influence of spermine on chlorophyll biosynthesis allowed us to determine approximately and to discuss the likely role of spermine in the biosynthesis of photosynthetic pigments.

Photoreactions of riboflavin with spermine and their role in tryptophan photoconsumption induced by riboflavin

The influence of spermine (Spr) on riboflavin (Rb) and riboflavin—tryptophan (Rb-Trp) solutions irradiated at 450 nm under aerobic and anaerobic conditions was studied. In both systems, it is shown that Spr interacts with 3Rb probably giving spermine-derived and flavin-derived radicals. In the presence of oxygen, modified Rb is regenerated with the simultaneous production of the active oxygen species O 2 .− and/or HO 2 .. The addition of Spr to the Rb-Trp system under aerobic conditions leads to a small protection of Trp consumption, although oxygen consumption is increased. This small protection arises as a consequence of competitive interaction of Trp and Spr with 3Rb. The competition of Spr and oxygen for 3Rb could also explain the autocatalytic oxygen consumption observed in the presence of the amine. Under anaerobic conditions Spr produces a larger protection of Trp consumption, which is compatible with the larger Rb triplet lifetime expected under these conditions.

Responsibility of tRNA Ile for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA Ile and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA Ile but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the V max and K m values for rat liver tRNA Ile. The K m value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA Ile tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PP i-ATP exchange in the presence of rat liver tRNA Ile, an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA Ile was determined and compared with that of E. coli tRNA Ile. Differences in nucleotide sequences of the two tRNAs Ile were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V 1 digestion of the 3′- 32P-labeled rat liver tRNA Ile showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg 2+. The influence of spermine on the ribonuclease V 1 digestion of E. coli tRNA Ile was different from that of rat liver tRNA Ile. The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.

Different binding modes of spermine to A-T and G-C base pairs modulate the bending and stiffening of the DNA double helix.

The influence of base composition (and sequence) on the process of interaction between synthetic polynucleotides and spermine, has been investigated using ultraviolet (including second derivative) spectroscopy, and electric dichroism. Different binding modes of spermine to poly(dG-dC) as compared to A-T containing polynucleotides, were evidenced. An interaction with the N7 and O6 of guanine is probably partially involved in the former case while simple electrostatic interaction with the phosphate groups would dominate in the latter. In the intermediate binding range (spermine over DNA phosphate molar ratios Sp/P of the order of 0.1 to 0.2), the complexes with poly(dA).poly(dT) and those with poly(dA-dT) displayed an important contribution of a permanent dipole moment to the orientation mechanism, as detected by the application of bipolar pulses in electric dichroism experiments. Just prior to precipitation (at Sp/P slightly larger than 0.3), these polynucleotides show electric dichroism and relaxation times characteristics corresponding to toroidal particles formation resulting from a bending of their chains. This implies asymmetric binding to phosphate sites on A-T containing polynucleotides. At low Sp/P ratios, spermine induced a stiffening of poly(dG-dC). No influence of spermine on the orientation mechanism of this polynucleotide was detected for Sp/P values ranging from zero to 0.35. The spermine-induced bending of A-T rich regions thus appears to be essential for DNA condensation into toroidal particles.

Different binding modes of spermine to A-T and G-C base pairs modulate the bending and stiffening of the DNA double helix

The influence of base composition (and sequence) on the process of interaction between synthetic polynucleotides and spermine, has been investigated using ultraviolet (including second derivative) spectroscopy, and electric dichroism. Different binding modes of spermine to poly(dG-dC) as compared to A-T containing polynucleotides, were evidenced. An interaction with the N7 and O6 of guanine is probably partially involved in the former case while simple electrostatic interaction with the phosphate groups would dominate in the latter. In the intermediate binding range (spermine over DNA phosphate molar ratios Sp/P of the order of 0.1 to 0.2), the complexes with poly(dA).poly(dT) and those with poly(dA-dT) displayed an important contribution of a permanent dipole moment to the orientation mechanism, as detected by the application of bipolar pulses in electric dichroism experiments. Just prior to precipitation (at Sp/P slightly larger than 0.3), these polynucleotides show electric dichroism and relaxation times characteristics corresponding to toroidal particles formation resulting from a bending of their chains. This implies asymmetric binding to phosphate sites on A-T containing polynucleotides. At low Sp/P ratios, spermine induced a stiffening of poly(dG-dC). No influence of spermine on the orientation mechanism of this polynucleotide was detected for Sp/P values ranging from zero to 0.35. The spermine-induced bending of A-T rich regions thus appears to be essential for DNA condensation into toroidal particles.

The influence of spermine on the structural dynamics of yeast tRNA Phe

A tRNA Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T 1–T 3) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T 1 and T 2, and a slow one (100–1000 ms) between T 2 and T 3. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T 3, as does Mg 2+, but the final ratio [ T 2] [ T 1] obtained with spermine is higher than with Mg 2+, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.

Mechanism of aminoacylation of tRNA: Influence of spermine on the kinetics of aminoacyl-tRNA synthesis by isoleucyl- and valyl-tRNA synthetases from Mycobacterium smegmatis

Isoleucyl-tRNA synthetase has been purified to homogeneity from Mycobacterium smegmatis. The influence of spermine on the kinetics of valyl-tRNA and isoleucyl-tRNA formation has been investigated by Cleland's method (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104–137, 173–187, 188–196). The results suggest that in the presence of spermine and suboptimal concentration of Mg 2+, the formation of valyl-tRNA and isoleucyl-tRNA follows a sequential ∗ ∗ In Cleland's nomenclature ‘sequential’ mechanism is equivalent to the ‘concerted’ mechanism used by others for aminoacylation. The ‘ping-pong’ mechanism of Cleland is equivalent to ‘step-wise’ or ‘sequential’ mechanism as used for aminoacylation. In the summary and results, Cleland's nomenclature is used; in discussion, a general one is used. mechanism. In the presence of an optimal concentration of Mg 2+, both valyl-tRNA and isoleucyl-tRNA formation proceeds by a ping-pong mechanism. However, in the presence of spermine and optimal concentrations of Mg 2+, valyl-tRNA formation follows the ping-pong mechanism while isoleucyl-tRNA formation follows the sequential mechanism.