Protein synthesis in rabbit reticulocytes XXII+: a heat stable dialyzable factor (EIF-I*) modulates Met-tRNAf binding to EIF-1.

The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNA_f·EIF-1·GTP in the absence of Mg⁺⁺ and the preformed complex is stable to Mg⁺⁺. However, with homogeneous preparations of EIF-1, addition of Mg⁺⁺ during the initial formation of the ternary complex strongly inhibits the complex formation.A heat stable dialyzable factor (EIF-1¹) which mostly remains associated with the high molecular weight protein complex, EIF-2 (TDF) during purification of the peptide chain initiation factors, has been purified using a phenol extraction procedure. EIF-1¹ restores the Met-tRNA_f binding activity of EIF-1 in the presence of 1 mM Mg⁺⁺; in the presence of EIF-1¹, Met-tRNA_f binding by EIF-1 shows a sharp Mg⁺⁺ optimum around 1 mM. EIF-1¹ is heat stable, alkali stable, dialyzable and pronase sensitive. The same EIF-1¹ preparation also strongly inhibits Met-tRNA_f binding to EIF-1 in the absence of Mg⁺⁺ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system.     

Chelation of divalent cations by lomofungin: role in inhibition of nucleic acid synthesis

Lomofungin inhibition of yeast growth and RNA synthesis is prevented by Cu⁺⁺ or Zn⁺⁺ ions which chelate with the antibiotic and prevent its uptake by the cells. EDTA potentiates the inhibition. Mg⁺⁺ ions do not protect $\text{in vivo}$ or against the inhibition of purified bacterial RNA and DNA polymerases. Lomofungin prevents formation of the RNA polymerase. DNA initiation complex, probably by chelation with the firmly bound Zn⁺⁺ of the enzyme.     

Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

Effect of spermine on peptide-bond formation,catalyzed by ribosomal peptidyltransferase

The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli.In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg⁺⁺ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg⁺⁺ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg⁺⁺, or was abolished at 10 mM Mg⁺⁺ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome.In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.     

Protein Synthesis in Cell-Free Preparations of Crithidia fasciculata*

SYNOPSIS. Cell-free preparations from Crithidia jasciculata carried out protein synthesis as measured by ¹⁴C-leucine uptake (optimum ～ 10 mM Mg⁺⁺) and poly U-directed ¹⁴C-phenylalanine uptake (optimum ～ 16 mM Mg⁺⁺). Characteristics of the system and sucrose density-gradient patterns of ribosomes were investigated. The charging and transfer reactions—the 2 main steps in protein synthesis—were inhibited by stilbamidine, hydroxystilbamidine, pentamidine, quinapyramine (Antrycide), and suramin.     

Polyamine als Stimulatoren von Genaktivitäten Eine autoradiographische Studie an Riesenchromosomen von Chironomus thummi

Zusammenfassung Inaktive Riesenchromosomen der Speicheldrüsen ausgewachsener Chironomus thummi-Larven zeigten nach 60 min Inkubation mit 0,05 N Putrescin-, Spermidin-oder Spermin-Lösung erneute Syntheseaktivitäten, die sich im Einbau von radioaktiv markiertem Uridin autoradiographisch zu erkennen geben. Diese Beobachtung wird im Zusammenhang mit der bekannten genaktivierenden Wirkung von Mg⁺⁺ diskutiert. Auf der Basis zahlreicher biochemisch analoger Wirkungen von Polyaminen und Mg⁺⁺ wird ein ursprünglich für die genaktivierende Wirkung von Mg⁺⁺ postuliertes Modell auf Polyamine übertragen.

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Stimulation of gene activities by polyamines an autoradiographic study with giant chromosomes of Chironomus thummi

Summary Inactive salivary gland giant chromosomes of full grown Chironomus thummi larvae showed after 60 min of incubation in 0.05 N putrescine, spermidine or spermine solutions autoradiographically demonstrable incorporation of radioactiv labelled uridine caused by a reactivation of RNA synthesis. This observations is discussed in relation to the known gene activating effect of Mg⁺⁺, and, since polyamines and Mg⁺⁺ have numerous effects in common, it is suggested that polyamines affect gene activity in a way similar to Mg⁺⁺.

     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Effect of mersalyl on mitochondrial Mg++ flux

The mercurial mersalyl has little effect either on rapid Mg⁺⁺ binding by isolated rat liver mitochondria or on the total Mg⁺⁺ content of these organelles measured after 0.75 min of incubation at 20°C. The data do not support the previous suggestion that the increased permeability to K⁺ of mitochondria treated with mersalyl results from release of endogenous Mg⁺⁺. An increased pH-dependence of unidirectional Mg⁺⁺ flux into respiring rat liver mitochondria is suggested to arise indirectly from inhibition by mersalyl of pH shifts associated with exchanges of endogenous phosphate. In addition, mersalyl appears to have a stimulatory effect on Mg⁺⁺ influx. Mersalyl also increases the average rate of unidirectional efflux of endogenous Mg⁺⁺. The stimulatory effects of mersalyl on Mg⁺⁺ flux are similar to, although quantitatively less than, the previously reported effects of mersalyl on mitochondrial K⁺ flux.     

The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland

The stimulatory effect of spermidine on the translation of poly(A)⁺ mRNA from lactating mouse mammary glands in a wheat germ system was studied. Spermidine stimulated total polypeptide synthesis about 2.5-fold relative to that occurring in the presence of an optimal concentration of Mg²⁺ alone. The size and the number of polysomes were about 1.6-times larger in the presence of spermidine than in its absence. A similar magnitude of increase in peptide chain initiation, 1.4-fold, was found when the extent of peptide chain initiation was measured by determining the residual polypeptide synthesis subsequent to the addition of inhibitor(s) of peptide chain initiation to the in vitro translation system with or without spermidine at various times of the incubation. Time-course study of the release of polypeptide from polysomes showed that spermidine stimulated this process to a much greater extent than peptide chain initiation, indicating that the polyamine also increases the rate of peptide chain elongation. The extent of stimulation of peptide chain elongation by spermidine was estimated to be about 1.5-fold when the disappearance of isotope-labeled nascent peptides from polysomes was measured by pulse-chase experiments. These results indicate that spermidine stimulates the cell-free translation of mammary mRNA by increasing the rates of both initiation and elongation of polypeptide synthesis to almost the same extent. The polyamine also reduced the relative amount of incomplete polypeptides, thereby increasing the yield of full-length translational products.     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

Protein synthesis in rabbit reticulocytes XX: A supernatant factor (TDI) inhibits ternary complex (Met-tRNAf·EIF-1·GTP) dissociation and Met-tRNAf binding to 40S ribosomes

A protein synthesis initiation inhibitor, TDI has been partially purified from the reticulocyte cell-supernatant. TDI inhibits the dissociation of the ternary complex, Met-tRNA_f·EIF-1·GTP and also Met-tRNA_f binding to 40S ribosomes. TDI inhibition requires Mg⁺⁺ and the inhibition is also observed when GTP is replaced by a non-hydrolyzable analog, GMP-PNP.     

Effect of different water salinity levels and organic matter application on the composition of water soluble and exchangeable bases in a brackishwater fish pond soil

Behaviour of different water soluble and exchangeable bases in a brackishwater fish pond soil was studied under four levels of water salinity, in combination with and without organic matter application. The results showed average content of water soluble bases to increase with increase in water salinity. The bases were dominated by Na⁺ followed by Mg⁺⁺, Ca⁺⁺ and K⁺ in decreasing order. SAR values of water increased with increase in water salinity and decreased slightly on organic matter treatment.Total content of exchangeable bases in soils was fairly high and was dominated by Ca⁺⁺ and Mg⁺⁺, followed by Na⁺ and K⁺ respectively. Amount of exchangeable Ca⁺⁺ + Mg⁺⁺ decreased while that of Na⁺ increased with increase in water salinity levels. Amount of exchangeable K⁺ did not show any appreciable change. Application of organic matter tended to increase the exchangeable Ca⁺⁺ + Mg⁺⁺ content and decrease the amount of exchangeable Na⁺ in the soil, while exchangeable K⁺ content remained practically unaffected due to organic matter treatment.Formed part of a Ph.D. thesis submitted to Bidhan Chandra Agricultural University, India in 1978Formed part of a Ph.D. thesis submitted to Bidhan Chandra Agricultural University, India in 1978     

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux

Effects on Mg⁺⁺ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K⁺ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K⁺ flux into respiring mitochondria, also stimulates Mg⁺⁺ influx. The K⁺ analog Ba⁺⁺, when taken up into the mitochondrial matrix, inhibits influx of both K⁺ and Mg⁺⁺. The effect on Mg⁺⁺ influx is seen only if Mg⁺⁺, which blocks Ba⁺⁺ accumulation, is added after a preincubation with Ba⁺⁺. Thus the inhibition of Mg⁺⁺ influx appears to require interaction of Ba⁺⁺ at the matrix side of the inner mitochondrial membrane. Added Ba⁺⁺ also diminishes observed rates of Mg⁺⁺ efflux but not K⁺ efflux. This difference may relate to a higher concentration of Ba⁺⁺ remaining in the medium in the presence of Mg⁺⁺ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexylcarbodiimide (DCCD), under conditions which result in an increase in the apparentK _m for K⁺ of the K⁺ influx mechanism, results in inhibition of Mg⁺⁺ influx from media containing approximately 0.2 mM Mg⁺⁺. The inhibitory effect of DCCD on Mg⁺⁺ influx is not seen at higher external Mg⁺⁺ (0.8 mM). This dependence on cation concentration is similar to the dependence on K⁺ concentration of the inhibitory effect of DCCD on K⁺ influx. Although mitochondrial Mg⁺⁺ and K⁺ transport mechanisms exhibit similar reagent sensitivities, whether Mg⁺⁺ and K⁺ share common transport catalysts remains to be established.Abbreviations used: DCCD, dicyclohexylcarbodiimide; PheAsO, phenylarsine oxide.     

The participation of phosphate in the formation of a carrier for the transport of Mg++ and Mn++ ions into yeast cells

During the absorption of phosphate by yeast, the cells acquire the capacity to absorb Mn⁺⁺ and Mg⁺⁺, a capacity which is retained even after phosphate is no longer present in the medium. Cells pretreated with phosphate and then washed, slowly lose their ability to absorb Mn⁺⁺, the rate of loss depending on the temperature and on the metabolic state. The fermentation of sugars induces a very rapid loss of absorptive capacity, whereas the respiration of ethyl alcohol, lactate, or pyruvate has little effect. Inhibitor studies with sodium acetate, redox dyes, and arsenate, reveal parallel effects on Mn⁺⁺ absorption, and on phosphate absorption. It is concluded that the synthesis of a carrier for the transport of Mg⁺⁺ and Mn⁺⁺ involves a phosphorylation step closely coupled with reactions involved in the absorption of phosphate.     

The response to oxidative stress induced by magnesium deficiency in kidney bean plants

To understand the plant response to oxidative stresses, we studied the influence of magnesium (Mg⁺⁺) deficiency on the formation of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and protease activity in kidney bean plants. The expression pattern of proteins under Mg⁺⁺ deficiency also was examined via two-dimensional electrophoresis. The formation of H₂O₂ and MDA increased in the primary leaves of plants grown in a nutrient solution deficient in Mg⁺⁺. Protease activity in Mg⁺⁺-deficient plants was also higher than in those grown with sufficient Mg⁺⁺. The expression pattern of the proteins showed that 25 new proteins were generated and 64 proteins disappeared under Mg⁺⁺-deficient conditions. Therefore, a deficiency in Mg⁺⁺ may cause oxidative stress and a change in protein expression. Some of these proteins may be related to the oxidative stress induced by Mg⁺⁺ deficiency.     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.     

The effect of Mg ++ on the conformation of the Ca ++ -binding component of troponin

Mg⁺⁺ like Ca⁺⁺ induces a conformational change in the Ca⁺⁺-binding component of troponin. However, this change is only 36 % of the change in fluorescence intensity and 80 % of the change in optical rotation induced by Ca⁺⁺. The apparent binding constant of Mg⁺⁺ to the Ca⁺⁺-binding component is 5 × 10³ M⁻¹, much smaller than that of Ca⁺⁺. Circular dichroism measurements show that these changes are simple helix-coil transitions. Unlike the Ca⁺⁺-induced conformational change, the Mg⁺⁺-induced change cannot be propagated to other muscle proteins, and therefore has no physiological meaning.     

The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.