Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3′,4′-didemethoxy-3′,4′-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl₂ but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl₂. Complex formation between the parent quinones and Mg⁺² was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg⁺². These observations suggest a possible role of Mg⁺² chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.     

Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

Ammonia removal from anaerobically digested dairy manure by struvite precipitation

Ammonia is one of the most important contaminants impairing the quality of water resources. When this is considered along with the fact that the global demand for nitrogenous fertilizers is in constant rise, the need for recovery as well as removal of nitrogen is well justified. Crystallization of N and P in the form of struvite (MgNH₄PO₄·6H₂O), which is a slow releasing and valuable fertilizer, is one possible technique for this purpose. This study investigated the removal of NH₄⁺ through struvite precipitation from the effluents of one- (R1) and two-phase (R2) anaerobic reactors digesting dairy manure. To force the formation of struvite in the anaerobic reactor effluents, Mg²⁺ ion was added by using both Mg(OH)₂ and MgCl₂·6H₂O. To prevent the effect of different total phosphorus (TP) concentration in the effluents of R1 and R2, as well as to not limit the formation of struvite, an excess amount of PO₄³⁻ (0.14 M) was added in the form of Na₂HPO₄. Different stoichiometric Mg²⁺:NH₄⁺:PO₄³⁻ ratios were tested to determine the required Mg²⁺ concentrations for maximum NH₄⁺ removal by keeping NH₄⁺:PO₄³⁻ ratio constant for the effluents of reactors R1 and R2. The results revealed that very high NH₄⁺ removal efficiencies (above 95%) were possible by adding Mg²⁺ ions higher than 0.06 M concentration in the effluents from reactors R1 and R2. It was also observed that the initial pH adjustment to 8.50 using NaOH did not result in any significant increase in the removal of NH₄⁺ and the removal of NH₄⁺ in the reactors treated with MgCl₂·6H₂O was higher than those treated with Mg(OH)₂ for the same Mg²⁺ concentration.     

Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens

Mg²⁺ can potentially influence bacterial adhesion directly through effects on electrostatic interactions and indirectly by affecting physiology-dependent attachment processes. However, the effects of Mg²⁺ on biofilm structure are largely unknown. In this study, Pseudomonas fluorescens was used to investigate the influence of Mg²⁺ concentration (0, 0.1 and 1.0 mM MgCl₂) on biofilm growth. Planktonic and attached cells were enumerated (based on DAPI staining) while biofilm structures were examined via confocal laser scanning microscopy and three-dimensional structures were reconstructed. Mg²⁺ concentration had no influence on growth of planktonic cells but, during biofilm formation, Mg²⁺ increased the abundance of attached cells. For attached cells, the influence of Mg²⁺ concentration changed over time, suggesting that the role of Mg²⁺ in bacterial attachment is complex and dynamic. Biofilm structures were heterogeneous and surface colonization and depth increased with increasing Mg²⁺ concentrations. Overall, for P. fluorescens, Mg²⁺ increased initial attachment and altered subsequent biofilm formation and structure.     

The (Na + K)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl2

Na⁺-dependent ADP/ATP exchange activity, of a (Na⁺ + K⁺)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of ¹⁴C into ATP during incubations with unlabeled ATP and [¹⁴C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl₂ concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg²⁺, Mg · ATP, and Mg · ADP present, with a range of MgCl₂, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg²⁺, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg²⁺, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg²⁺, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.     

Effect Of Glutathione On The Redox Transitions Of Naphthohydroquinone Derivatives Formed During Dt-Diaphorase Catalysis

The oxidation of GSH coupled to the redox transitions of 1, Cnaphthoquinone derivatives during DT-diaphorase catalysis was examined. The quinones studied included 1,4-naphthoquinone and its dimethoxy-and hydroxy derivatives and were selected according to their different ability to undergo nucleophilic addition with GSH and the dual effect of superoxide dismutase on hydroquinone autoxidation

GSH was oxidized to GSSG during the redox transitions of the above quinones, regardless of their substitution pattern. This effect was accompanied by an increase of total O₂ consumption, indicating the ability of GSH to support quinone redox cycling. The values for the relationship [O₂]_consumed[GSSG]_formde were, with every quinone examined, above unity. thus pointing to the occurrence of autoxidation reactions other than those involved during GSSG formation

These results are discussed in terms of the functional group chemistry of the quinones and the ther-modynamic properties of the reactions involved in the reduction of the semi- to the hydro-quinone by GSH     

Intersystem exciton transfer in isolated chloroplasts

The following arguments in favor of exciton transfer between the two photosystems are presented:

1. (1) MgCl₂ (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl₂ addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg²⁺.

2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I.

3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl₂, the values of rate constants of Photosystem II deactivation were calculated.

4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl₂ addition. When this transfer is decreased by MgCl₂ the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained.

A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl₂ decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.     

Release of [H]GABA evoked by glutamate agonists from hippocampal slices: effects of dithiothreitol and glutathione

The effects of dithiothreitol (DTT) and, reduced (GSH) and oxidized (GSSG), glutathione on the release of [³H]GABA evoked by glutamate and its agonists were studied in rat hippocampal slices. DTT had no effect on the basal release of [³H]GABA but it enhanced and prolonged the glutamate agonist-evoked release. This effect was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801), a noncompetitive NMDA antagonist, and blocked by Mg²⁺ ions. It was only slightly attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, and not affected by -(+)-2-amino-3-phosphonopropionate ( -AP3), a selective antagonist of the metabotropic glutamate receptor. The effect of DTT on the NMDA-evoked release of GABA was only slightly affected by extracellular Ca²⁺ but completely blocked by verapamil even in the absence of Ca²⁺. GSH and GSSG attenuated or abolished the effects of DTT on the agonist-induced release of [³H]GABA. The results imply that the enhanced and prolonged release of GABA evoked by the coexistence of DTT and excitatory amino acids and attenuated by endogenous GSH and GSSG is a consequence of sustained activation of the NMDA receptor-governed ionophores, which contain functional thiol groups. DTT, GSH and GSSG may regulate the redox state and accessibility of these groups. In addition to the influx of extracellular Ca²⁺, DTT mobilizes Ca²⁺ from intracellular pools distinct from those regulated by metabotropic glutamate receptors.     

Sulfane-Activated Reduction of Cytochrome c by Glutathione

The inorganic sulfane tetrathionate (^-O₃SSSSO₃^-) resembles glutathione trisulfide (GSSSG) in that it remarkably activates the reduction of cytochrome c by GSH, both under aerobic and anaerobic conditions. These observations can be explained by the formation of the persulfide GSS^-, due to nucleophilic displacements of sulfane sulfur. The GSS^- species has previously been proposed to act as a chain carrier in the catalytic reduction of cytochrome c, and perthiyl radicals GSS·, formed in the reduction step, were thought to recycle to sulfane via dimerization to GSSSSG.² The present study provides some arguments in favour of a chain mechanism involving the GSS· + GS^- ⇄ (GSSSG)^- equilibrium and sulfane regeneration by a second electron transfer from (GSSSG)· ^- to cytochrome c.

Thiosulfate sulfurtransferase (rhodanese) is shown to act as a cytochrome c reductase in the presence of thiosulfate and GSH, and again the generation of GSS^- can be envisaged to explain this result.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts

The effects of divalent metal ions on the yields of chlorophyll a fluorescence were investigated in isolated spinach chloroplasts at room and liquid nitrogen temperatures. Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺ increased the yields of fluorescence emission at 684 and 695 nm from pigment system II and decreased that at 735 nm from pigment system I. Al³⁺ showed similar but less significant effects on the fluorescence yields. Zn²⁺ and Cd²⁺ showed no significant effect on the fluorescence yields at concentrations lower than 5 mM.

In accordance with the results of our previous study concerning the effects of Mg²⁺ on the excitation transfer in the chloroplasts, it was concluded that ions of alkaline earths and manganese suppress the excitation transfer from bulk chlorophylla of pigment system II to that of pigment system I.     

The effect of hydroxylamine on microsomal ATPase

The (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase, but not the Mg²⁺-ATPase, is irreversibly inhibited when turtle bladder microsomes were incubated with hydroxylamine.

The Mg²⁺-dependent or the (Mg²⁺ + Na⁺)-dependent phosphorylation of ADP by the phospho-protein (the exchange reaction) is reversibly inhibited when the microsomes are incubated with hydroxylamine.

The Na⁺-induced increment of ³²P-labelling of microsomes previously incubated with [λ-³²P]ATP is completely eliminated by hydroxylamine, but the Mg²⁺-dependent ³²P-labelling of such microsomes is unaffected by hydroxylamine.

It is concluded that the phospho-enzyme formed during the Mg²⁺-dependent hydrolysis does not contribute to the Mg²⁺-dependent exchange reaction. Instead, the phosphoenzyme formed during the (Na⁺ + K⁺)-stimulated hydrolysis is apparently the only substance which phosphorylates ADP in the exchange reaction, even in the absence of Na⁺ and/or K⁺.

The hydroxylamine-sensitive nature of the sodium form of the phospho-enzyme in the (Na⁺ + K⁺)-stimulated ATPase sequence is consistent with the existence of an enzyme-acyl-phosphate bond of high internal energy with respect to that of ADP.

On the other hand, the hydroxylamine-resistant nature of the phospho-enzyme in the Mg²⁺-ATPase sequence suggests the existence of a non-acyl type of enzyme phosphate bond with low internal energy relative to that of ADP.     

Synthetic quinones influencing herbicide binding and Photosystem II electron transport. The effects of triazine-resistance on quinone binding properties in thylakoid membranes

We have analyzed the binding of synthetic quinones and herbicides which inhibit electron transport at the acceptor side of Photosystem II (PS II) of the photosynthetic electron-transport chain in thylakoid membranes. These data show that quinones and PS II-directed herbicides compete for binding to a common binding environment within a PS II region which functions as the Q⁻ / PQ oxidoreductase. We observed that (1) synthetic quinones cause a parallel inhibition of electron transport and [¹⁴C]herbicide displacement, and (2) herbicide binding is affected both by the fully oxidized and fully reduced form of a quinone. Quinone function and inhibitor binding were also investigated in thylakoids isolated from triazine-resistant weed biotypes. We conclude the following. (1) The affinity of the secondary accepting quinone, B, is decreased in resistant thylakoids. (2) The observation that the equilibrium concentration of reduced Q after transferring one electron to the acceptor side of PS II is increased in resistant as compared to susceptible chloroplasts may be explained both by a decrease in the affinity of PQ for the herbicide / quinone binding environment, and by a decrease of the midpont redox potential of the B / B⁻ couple. (3) The binding environment regulating quinone and herbicide affinity may be divided roughly into two domains; we suggest that the domain regulating quinone head-group binding is little changed in resistant membranes, whereas the domain-regulating quinone side-group binding (and atrazine) is altered. This results in increased inhibitory activity of tetrachloro-p-benzoquinone and phenolic herbicides, which are hypothesized to utilize the quinone head-group domain. The two domains appear to be spatially overlapping because efficient atrazine displacement by tetrachloro-p-benzoquinone is observed.     

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

The Effect of Divalent Cations and Core Polymerase in the Abortive Initiation by Escherichia Coli Dna-Dependent Rna Polymerase Using Phosphonate Dinucleotide Primers

The effect of divalent Mg²⁺ and Mn²⁺ cations on the elongation of ApU, UpA and their 3'-O- and 5'-O-phosphonylmethyl analogues by RNA polymerase holoenzyme to the corresponding trinucleo-tides on a poly(dA-dT) template was investigated. In contrast to Mg^z+ ions, Mn²⁺ ions enhance abortive trinucleotide synthesis. This effect is more pronounced with phosphonylmethyl analogues. The core enzyme cannot catalyze the elongation of either (2'-5') UpA or phosphonylmethyl analogues. The localization of the divalent cation activator, as well as the role of the σ subunit at the catalytic centre of the holoenzyme, is discussed.     

Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

Molecular mechanisms of quinone cytotoxicity

Quinones are probably found in all respiring animal and plant cells. They are widely used as anticancer, antibacterial or antimalarial drugs and as fungicides. Toxicity can arise as a result of their use as well as by the metabolism of other drugs and various environmental toxins or dietary constituents. In rapidly dividing cells such as tumor cells, cytotoxicity has been attributed to DNA modification. However the molecular basis for the initiation of quinone cytotoxicity in resting or non-dividing cells has been attributed to the alkylation of essential protein thiol or amine groups and/or the oxidation of essential protein thiols by activated oxygen species and/or GSSG. Oxidative stress arises when the quinone is reduced by reductases to a semiquinone radical which reduces oxygen to superoxide radicals and reforms the quinone. This futile redox cycling and oxygen activation forms cytotoxic levels of hydrogen peroxide and GSSG is retained by the cell and causes cytotoxic mixed protein disulfide formation. Most quinones form GSH conjugates which also undergo futile redox cycling and oxygen activation. Prior depletion of cell GSH markedly increases the cell's susceptibility to alkylating quinones but can protect the cell against certain redox cycling quinones. Cytotoxicity induced by hydroquinones in isolated hepatocytes can be attributed to quinones formed by autoxidation. The higher redox potential benzoquinones and naphthoquinones are the most cytotoxic presumably because of their higher electrophilicty and thiol reactivity and/or because the quinones or GSH conjugates are more readily reduced to semiquinones which activate oxygen.     

Mechanism of the Mg-facilitated specific cleavage of the terminal phosphoryl group of adenosine 5′-triphosphate

The exchange rate constant between free Mg²⁺ and Mg²⁺ bound to adenosine 5′-triphosphate (ATP) was determined at various temperatures from the ³¹P-NMR spectra of ATP in the absence and presence of Mg²⁺. The activation free energy of this exchange reaction showed that Mg²⁺ binds asymmetrically to the β- and γ-phosphoryl groups and that it coordinates with the β-phosphoryl group more tightly than with the γ-phosphoryl group of ATP. On binding, Mg²⁺ becomes located closer to the β-phosphoryl group. This asymmetric location of Mg²⁺ weakens the chemical bond of the terminal bridged phosphoryl group, thus causing specific cleavage of this group. This mechanism was confirmed by an ab initio molecular orbital calculation, and by experiments on the stability of ATP in aqueous solution.