Interaction of yeast arginyl-tRNA synthetase and aspartyl-tRNA synthetase with Blue-dextran Sepharose : assignment of the Blue-Dextran Binding site on the synthetases

Yeast arginyl-tRNA synthetase and aspartyl-tRNA synthetase like nucleotidyl transferases previously investigated interact with the Blue-Dextran-Sepharose affinity ligand through their tRNA binding domain: the enzymes are readily displaced from the affinity column by their cognate tRNAs but not by ATP or a mixture of ATP and the cognate amino acid in contrast to other aminoacyl-tRNA synthetases. In the absence of Mg⁺⁺, the arginyl-tRNA synthetase can be dissociated from the column by tRNA^Asp and tRNA^Phe which have been shown to be able to form a complex with the synthetase, but in presence of Mg⁺⁺ the elution is only obtained by the specific tRNA.The procedure described here can thus be used: (i) to detect polynucleotide binding sites in a protein; (ii) to estimate the relative affinities of different tRNAs for a purified synthetase; (iii) to purify an aminoacyl-tRNA synthetase by selective elution with the cognate tRNA.     

Cation activation of desoxyribonuclease

The activation of desoxyribonuclease on desoxyribonucleate, known to occur with Mg⁺⁺ and Mn⁺⁺, has been shown to occur equally well with Co⁺⁺, to nearly the same extent with Fe⁺⁺, and to a lesser extent with Ca⁺⁺, Ba⁺⁺, Sr⁺⁺, Ni⁺⁺, Cd⁺⁺, and Zn⁺⁺. The conditions under which the optimal activation is revealed vary among these ions. Thus, Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ may show marked activation under conditions in which Fe⁺⁺ is nearly ineffective. Since too high a concentration of an ion may be as ineffective as too little, concentration-activation curves were determined for each ion. Per micromole of nucleic acid phosphorus, the optimal effective amount of each ion in micromoles is as follows: Mg⁺⁺ 3, Mn⁺⁺ 3, Co⁺⁺ 3, Fe⁺⁺ 0.3, Ni⁺⁺ 0.3, Ba⁺⁺ 1.7, Ca⁺⁺ 3, Sr⁺⁺ 3, Zn⁺⁺ 0.3, and Cd⁺⁺ 0.3.The optimum pH for the activation with Mg⁺⁺, Co⁺⁺, and Ca⁺⁺ is about 6.5, that with Fe⁺⁺ is at 5.7, while Mn⁺⁺ shows two optima at pH 6.8 and 8.0.Experiments conducted in Pyrex and in quartz vessels showed the same results, and indicated that there was no activation of desoxy-ribonuclease in the absence of added salts.     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

Ion and pH gradients across the transport membrane of mitochondria following Mn ++ uptake in the presence of acetate

The electron paramagnetic resonance (EPR) spectrum of Mn⁺⁺ loaded mitochondria is affected by the presence of the permeant anion acetate (Ac^?) in the medium. The hyperfine sextet, shown earlier to have spectral characteristics like those expected of osmotically active Mn⁺⁺ in the matrix space, grows in intensity with increasing [Ac^?]. From estimates of mitochondrial water, the free internal [Mn⁺⁺] can be calculated. The gradient of free [Mn⁺⁺] across the inner mitochondrial membrane is believed to be at least 500:1 under conditions of high [Ac^?]. Since Mn⁺⁺ solubility is limited by [OH^?], it is possible to place an upper limit on the pH in the matrix space. The variation of free internal [Mn⁺⁺], as measured by EPR, with external pH indicates that the [H⁺] gradient is 1–1.5 pH units in the absence of permeant anions and considerably less in the presence of 100 mM acetate.     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis     

Effect of the anthraquinones emodin and physcion on availability of selected soil inorganic ions

The effects of emodin and physcion, two anthraquinones involved in Polygonum sachalinense allelopathy, were studied in soils amended with 5, 10 and 15 mg 100 g^-1 soil of each compound, and with their mixtures. Recovery of each compound and its effects on availability of soil Mn⁺⁺, Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺ and PO₄^3- were monitored. Total phenolic content of each soil was determined to study the role of degraded phenolics. Soil amended with 5 mg of emodin and physcion gave maximum recovery of the two anthraquinones. Soil amended with different amounts of two anthraquinones and their mixture had significant lower values for pH compared to control. In general, both anthraquinones affect availability of soil inorganic ions. Compared to unamended control soil, a significant decrease in Mn⁺⁺ availability, and an increase in Na⁺ and K⁺ availability when emodin and physcion were added to the soil was observed. A correlation among soil inorganic ions, relative recovery of two compounds, and total phenolic content of amended soils was observed. Results indicate that emodin and physcion influence the availability of soil inorganic ions. The significance of the effect of allelopathic compounds on soil inorganic ions is discussed.     

Continuous electron spin resonance detection of biochemical reactions of nucleoside triphosphates.

A technique of continuous recording of the kinetics of biochemical reactions of nucleoside triphosphates by means of an ESR (electron spin resonance) method is proposed. The technique is based on the differential ability of NTP (nucleoside triphosphates) and the products of their conversion to coordinate Mn²⁺ ions. Due to this fact the concentration of free (hydrated) Mn²⁺ ions changes in the course of the reaction and, consequently, the intensity of their ESR signal also changes. The proposed technique makes it possible to determine changes of concentration ?0.1% of the total free ion concentration. The technique was applied to observation of reactions catalyzed by RNA polymerase, alkaline phosphatase, and aminoacyl-tRNA synthetase.     

Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn⁺⁺ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg⁺⁺ or Mn⁺⁺. The apparent Km values of adenylate cyclase for Mn⁺⁺-ATP and Mg⁺⁺-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn⁺⁺-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.     

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.     

Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M_r 1.82×10⁶, 0.84×10⁶ (high-M_r aminoacyl-tRNA synthetase complexes) and 0.12–0.35×10⁶. In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82×10⁶ complex into the 0.84×10⁶ complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. K_M for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivationin vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M_r complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.     

23Na NMR investigation of the interaction between DNA and divalent metallic cations

The aim of this study is to follow the thermodynamic behaviour of Na⁺ ions, acting as natural counterions of DNA, in the presence of divalent metal ions, by using the²³Na NMR technique. With the help of the²³Na entropy of fluctuations concept introduced by Lenk, we propose the following decreasing sequence: Mg⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, and Cu⁺⁺, for the magnitude of divalent metal ions interactions with DNA phosphate sites.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Production of Alkaline Lipase by Corynebacterium paurometabolum, MTCC 6841 Isolated from Lake Naukuchiatal, Uttaranchal State, India

A moderately psychrophilic bacterium Corynebacterium paurometabolum MTCC 6841 (gram positive, short rod type) producing extracellular alkaline lipase was isolated from Lake Naukuchiatal, Uttaranchal, India. The bacterium was able to grow within a broad range of pH (5–10). Soyabean oil and olive oil served as the best carbon sources for lipase production. The bacterium preferred inorganic nitrogenous compounds, NaNO₃ and KNO₃, over organic nitrogenous compound for its growth. Maximum lipase production occurred at 25°C and 8.5 pH. The enzyme activity was found to be maximum at the same values of temperature and pH. The enzyme was reasonably stable in the presence of various organic solvents. No significant effect of Ca⁺, Cu⁺⁺, Fe⁺⁺, Na⁺, K⁺, Mg⁺⁺, Mn⁺, NH4⁺, Co⁺⁺ ions over enzyme activity was detected. Treatment with EDTA reduced the activity to nearly one half.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

d-Xylose isomerase requires manganese ions for its action, but l-arabinose isomerase has a less specific on metal requirement. l-Arabinose isomerase is activated by addition of Mn⁺⁺ or Co⁺⁺, less effectively by addition of Zn⁺⁺, Ca⁺⁺, Mg⁺⁺, Sr⁺⁺ or Cd⁺⁺. Moreover, manganese and potassium ions for d-xylose isomerase, and manganese and cobaltous ions for l-arabinose isomerase were also shown to have protective effect on respective enzymes against thermal inactivation.     

Nuclear DNA-dependent RNA polymerases of Ehrlich ascites tumor cells: two discrete -amanitin-sensitive forms

DNA-dependent RNA polymerase was solubilized from nuclei of ascites tumor cells by the standard techniques of ultrasonic treatment in 0.3 M ammonium sulfate, salt fractionation, and dialysis. Three discrete forms of RNA polymerase (I, II, III) were separated on DEAE-Sephadex A-25. Forms II and III were inhibited by α-amanitin, but no form was sensitive to rifampicin. Each form was more active with Mn⁺⁺ than with Mg⁺⁺ ions, more active with denatured than with native calf thymus DNA, and differed from the others with respect to optimal concentrations of (NH₄)₂SO₄, Mn⁺⁺ ions and DNA.     

Acid phosphatase fractions from various growth zones of broad bean root revealed by disc electrophoresis

Fractions of acid phosphate (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) were studied in extracts of segments from three growth zones of broad bean roots by means of electrophoresis in acrylamide gel. The azocoupling reaction with α-naphtyl phosphate was used for detection. The phosphatase activity was investigated in the range of pH 3·6–7·2. Altogether nine fractions moving towards the anode were revealed. Some fractions differed slightly in their pH optimum. The presence of Mg⁺⁺ in the incubation medium resulted in the activation of two fractions, Mn⁺⁺ showed activation of three fractions and inhibition of the rest of the fractions; the presence of Zn⁺⁺ resulted in a slight inhibition of all fractions. Between electrophoreograms of extracts of segments from the division zone and electrophoreograms of extracts of segments from the enlargement zone and from the maturation zone considerable quantitative differences were found with one fraction; proportions of the other fractions were approximately identical in electrophoreograms of all three growth zones. The response to the presence of Mg⁺⁺, Mn⁺⁺ and Zn⁺⁺ in the incubation medium as well as the pH optima of the individual fractions were identical for all three growth zones.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Effect of some metals and related metal-orgaic compounds on ALA-dehydratase activity of corn

Summary The activity of ALA-dehydratase from corn seedlings is affected by Mn⁺⁺, Fe⁺⁺, Pb⁺⁺, Cu⁺⁺, Zn⁺⁺ and Sn⁺⁴ ions, in vivo Mn⁺⁺ and Fe⁺⁺ are ativators while Pb⁺⁺ and Sn⁺⁴ are inhibitors; in vitro Cu⁺⁺ and Zn⁺⁺ are inhibitors. The kinetic parameters (V_max and K_M) support the hypothesis that Mn, Fe, Sn and Pb ions act on the biosynthesis of the enzyme and Zn and Cu ions on the enzyme-substrate affinity. Some related metal-organic compounds interrere in vivo on the ALA-dehydratase activity modifying the kinetic parameters, therefore the enzyme biogenesis and/or enzyme-sustrat affinity are affected.     

Studies on Bacterial Protease

The neutral protease of Bacillus amylosacchariticus was inactivated by low concentrations of several metal-chelating agents and the inactivated enzyme with EDTA restored its activity almost completely by the addition of Zn⁺⁺ or Co⁺⁺ and partially by Fe⁺⁺ or Mn⁺⁺, if these metal ions were added shortly after the EDTA-treatment. The native enzyme was found to contain 0.19% of zinc together with a significant amount of calcium. Parallel increase in specific activity and zinc content of enzyme preparation was observed throughout the purification procedure. The elution pattern of enzyme activity on a CM-cellulose column chromatography also completely coincided with that of protein-bound zinc. A zinc-free inactive enzyme was also reactivated by the addition of zinc or cobalt ions, clearly showing that the neutral protease of B. amylosacchariticus is a zinc mctalloenzyme.