The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin–EDTA combination and their effectiveness as antibiofilm agents

Aims:  To evaluate the antimicrobial activities of aspirin, EDTA and an aspirin-EDTA (A-EDTA) combination against Pseudomonas aeruginosa , Escherichia coli and Candida albicans in planktonic and biofilm cultures.
Methods and Results:  Minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) were determined using twofold broth microdilution and viable counting methods, respectively. Aspirin's recorded MIC values ranged from 1·2 to 2·7 mg ml⁻¹. Checkerboard assay demonstrated a synergism in antimicrobial activity upon combination. Aspirin's minimal biofilm eradication concentration values (MBEC) against the established biofilms ranged between 1·35 and 3·83 mg ml⁻¹. A complete eradication of bacterial biofilms was achieved after a 4-h treatment with the A-EDTA combination.
Conclusion:  Both aspirin and EDTA possess broad-spectrum antimicrobial activity for both planktonic and biofilm cultures. Aspirin used at the MBEC for 24 h was successful in eradicating P. aeruginosa , E. coli and C. albicans biofilms established on abiotic surfaces. Moreover, the exposure to the A-EDTA combination (4 h) effected complete bacterial biofilm eradication.
Significance and Impact of the Study:  There is a continuous need for the discovery of new antimicrobial agents. Aspirin and EDTA are 'nonantibiotic drugs', the combination of which can be used successfully to treat and eradicate biofilms established on abiotic surfaces.     

Cloning of <Emphasis Type="Italic">Escherichia coli</Emphasis> K12 xylose isomerase (glucose isomerase) gene and studying the enzymatic properties of its expression product

The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21(DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45°C and pH 6.8. The enzyme required Mg²⁺ ions as a cofactor. When Mg²⁺ and Co²⁺ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15–20%. Complete replacement of Mg²⁺ with Co²⁺ decreased the enzyme activity. In the presence of Ca²⁺ at concentrations comparable to the concentration of Mg²⁺, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca²⁺. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45°C and was completely inactivated after incubation at 65°C for 1 h.     

Sub-Minimum Inhibitory Concentrations of Ceftibuten Reduce Adherence of Escherichia coli to Human Cells and Induces Formation of Long Filaments

The minimal inhibitory concentrations (MICs) of an antibiotic are present for only a certain period of time, after which they become sub-inhibitory concentrations (sub-MICs). These sub-MICs are still active because they can interfere with the mechanism of bacterial adhesion, which is the first step in the sequence of events leading to infection. The purpose of the present study was to investigate the effects of sub-MICs of ceftibuten, a new third-generation cephalosporin, on the adhesion of Escherichia coli (E. coli) to human buccal cells. The degree of inhibition was maximal at 1/2 MIC and then gradually returned toward to the control values at 1/128 the MIC. The differences were statistically significant from 1/2 to 1/32 MIC. Since the MIC was 0.5 μg/ml, concentrations from 0.25 to 0.015 μg/ml significantly reduce bacterial adhesion. Ceftibuten also caused marked elongation of E. coli. These findings could help to explain the efficacy showed by ceftibuten in the treatment of respiratory and urinary tract infections when administered once daily.     

Highly effective, water-soluble, hemocompatible 1,3-propylene oxide-based antimicrobials: poly[(3,3-quaternary/PEG)-copolyoxetanes

This study focuses on the solution antimicrobial effectiveness of a novel class of copolyoxetanes with quaternary ammonium and PEG-like side chains. A precursor P[(BBOx-m)(ME2Ox)] copolyoxetane was prepared by cationic ring-opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) to give random copolymers with 14-100 (m) mol % BBOx. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m, as viscous liquids in 100% yield. BBOx/ME2Ox and C12/ME2Ox ratios were obtained by (1)H NMR spectroscopy. C12-m molecular weights (M(n), 3.5-21.9 kDa) were obtained from (1)H NMR end group analysis. DSC studies up to 150 °C showed only thermal transitions between -69 and -34 °C assigned to T(g) values. Antibacterial activity for the C12-m copolyoxetanes was tested by determining minimum inhibitory concentrations (MICs) against Gram(+) Staphylococcus aureus and Gram(-) Escherichia coli and Pseudomonas aeruginosa . MIC decreased with increasing C12 mol percent, reaching a minimum in the range C12-43 to C12-60. Overall, the antimicrobial with consistently low MICs for the three tested pathogenic bacteria was C12-43: (bacteria, MIC, μg/mL) E. coli (6), S. aureus (5), and P. aeruginosa (33). For C12-43, minimum biocidal concentration (MBC) to reach 99.99% kill in 24 h required 1.5× MIC for S. aureus and 2× MIC for E. coli and P. aeruginosa . At 5× MIC against a challenge of 10(8) cfu/mL, C12-43 kills ≥99% S. aureus , E. coli , and P. aeruginosa within 1 h. C12-m copolyoxetane cytotoxicity toward human red blood cells was low, indicating good prospects for biocompatibility. The tunability of C12-m copolyoxetane compositions, effective antimicrobial behavior against Gram(+) and Gram(-) bacteria, and promising biocompatibility offer opportunities for further modification and potential applications as therapeutic agents.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

Assessment of the susceptibility of the gram-negative rods isolated from hospitalized patients to cefoperazone/sulbactam

The susceptibility to cefoperazone/sulbactam of 197 strains of Gram-negative rods demonstrating an ESBL-positive phenotype was determined. The assortment of the investigated strains was as follows (numbers of strains are given in the brackets): E. cloacae (63), S. marcescens (46), K. pneumoniae (21), P. mirabilis (17), E. coli (9), P. vulgaris (8), P. aeruginosa (20) and A. baumanni (13). 83 strains from 197 were susceptible (42.1%). The MIC values were determined and the disc-diffusion method was performed. The susceptibilities among particular species were as follows (the order of data in the brackets is: % of the susceptible strains/MIC50/MIC90): E. cloacae (54.0/16/64), S. marcescens (23.9/64/> or = 128), K. pneumoniae (38.1/32/64), P. mirabilis (41.2/32/64), E. coli (44.4/32/32), P. vulgaris (75.0/8/32), P. aeruginosa (35.0/32/64), A. baumannii (46.2/32/64). Using disc-diffusion method, for 184 strains the difference between diameter of the inhibition zone around the disc with cefoperazone and the disc with cefoperazone/sulbactam was calculated. This difference amounted 5 mm or more in the case of 76.6% of the investigated strains. The results indicate that the comparison of the inhibition zones around cefoperazone and cefoperazone/sulbactam discs may be an additional method useful for phenotypic detection of ESBL producing organisms. These results highly correlated with results obtained by using analogous test with cefpirome and cefpirome/clavulanic acid (85.6% of concordance).     

Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg²⁺, Mn²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20°C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn²⁺ or Ca²⁺ was present in the buffered isozyme solution before the glucose titration, whereas Mg²⁺ caused the glucose binding to become noncooperative. Zn²⁺ and Cd²⁺ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, as well as in buffer alone, at both pH's. At the lower pH, Ca²⁺ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn²⁺ increased it only about 40% and Mg²⁺ not at all. Further, Ca²⁺ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg²⁺ and Mn²⁺ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca²⁺ effect on the cooperativity was negligible, while Mg²⁺ and Mn²⁺ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.     

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     

Influence of pH,Mg2+, and lipid composition on the aggregation state of the diatom FCP in comparison to the LHCII of vascular plants

In the present study, the influence of Mg²⁺ ions and low pH values on the aggregation state of the diatom FCP and the LHCII of vascular plants was studied. In addition, the concentration of thylakoid membrane lipids associated with the complexes was determined. The results demonstrate that the FCP, which contained a significantly higher concentration of the negatively charged lipids SQDG and PG, was less sensitive to Mg²⁺ and low pH values than the LHCII which was characterized by lower amounts of SQDG and a higher concentration of MGDG. High MgCl₂ concentrations and pH values below pH 6 induced significant changes of the absorption and 77K fluorescence emission spectra of the LHCII, indicating a strong aggregation of the light-harvesting complex. This aggregation was also visible as a pellet after centrifugation on a sucrose cushion. Although the FCP responded with changes of the absorption and fluorescence spectra to low pH and Mg²⁺ incubation, these spectral changes were less pronounced than those observed for the LHCII. In addition, the FCP complexes did not show a visible pellet after incubation with either low pH values or high Mg²⁺ concentrations. Only the combined action of Mg²⁺ and pH 5 led to FCP aggregates of a size that could be pelleted by centrifugation. The decreased sensitivity of FCP aggregation to Mg²⁺ and low pH is discussed with respect to the differences in the concentration of the lipids surrounding the FCP and LHCII and the different thylakoid membrane organizations of diatoms and vascular plants.     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

In vitro antibacterial activity of laboratory grown culture of Spirulina platensis

The hexane, ethyl acetate, dichloromethane, methanol extracts and spent media (extracellular substances) were tested in vitro for their antibacterial activity for which one Gram-positive bacterium (Staphylococcus aureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) were used as test organisms. The methanol extract showed more potent activity than other organic extracts, spent medium of the culture exhibited little activity against E. coli only. No inhibitory effect was found against Klebsiella pneumoniae.The broth microdilution assay gave minimum inhibitory concentrations (MIC) values ranging from 1 to 512 μg/ml. The MIC of methanol extract against S. aureus and E. coli were 128 μg/ml and 256 μg/ml, respectively.     

MIC Channels Are Inhibited by Internal Divalent Cations but Not ATP

TRPM7 channels are nonselective cation channels that possess a functional α-kinase domain. It has been proposed that heterologously expressed TRPM7 channels are activated (Runnels et al., 2001) or inhibited (Nadler et al., 2001) by dialyzing the cell with millimolar levels of ATP. The endogenous correlate of TRPM7 has been identified in T-lymphocytes and RBL (rat basophilic leukemia) cells and named MagNuM (for Mg²⁺-nucleotide-inhibited metal) or MIC (for Mg²⁺-inhibited cation). Here, we report that internal Mg²⁺ rather than MgATP inhibits this current. Cytoplasmic MgATP, supplied by dialysis at millimolar concentrations, effectively inhibits only when a weak Mg²⁺ chelator is present in the pipette solution. Thus, MgATP acts as a source of Mg²⁺ rather than a source of ATP. Using an externally accessible site within the pore of the MIC channel itself as a bioassay, we show that equimolar MgCl₂ and MgATP solutions contain similar amounts of free Mg²⁺, explaining the fact that numeric values of Mg²⁺ and MgATP concentrations necessary for complete inhibition are the same. Furthermore, we demonstrate that Mg²⁺ is not unique in its inhibitory action, as Ba²⁺, Sr²⁺, Zn²⁺, and Mn²⁺ can substitute for Mg²⁺, causing complete inhibition. We conclude that MIC current inhibition occurs simply by divalent cations.     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

INTERACTION OF LEUCINE, GLUCOSE, AND KETONE METABOLISM IN RAT BRAIN IN VITRO

Brain cortex slices from fed, 48 h and 120 h fasted rats were incubated and ¹⁴CO₂ was measured from (a) [U-¹⁴C]glucose (5 mm ) either alone or in the presence of l -lcucine (0.1 or 1 mm ), and (b) [U-¹⁴C]leucine or [l-¹⁴C]leucine at 0.1 or 1 mm with or without glucose (5 mm ). In other experiments, sodium dl -3-hydroxybutyrate (3-OHB) or acetoacetate (AcAc) at 1 or 5 mm were added in the above incubation mixture. The rate of conversion of [U¹⁴C]glucose to CO₂ was decreased 20% by leucine at 1 mm and 30–50% by 3-OHB at 1 or 5 mm but not by leucine at 0.1 mm . The effects of 3-OHB and of leucine (1 mm ) were not additive. The effects of leucine were similar in the fed and fasted rats. The rate of conversion of [U-¹⁴C]leucine or [l-^,4C]leucine to ¹⁴CO₂ at 0.1 mm and 1.0 mm was increased by glucose (35%) in the fed or fasted rats. Ketone bodies in the absence of glucose had no effect on leucine oxidation. However, the stimulatory effect of glucose on the rate of conversion of leucine to CO₂ was inhibited by 3-OHB at 5 mm . These results suggest that (a) leucine in increased concentrations (1 mm ) may reduce glucose oxidation by brain cortex while itself becoming an oxidative fuel for brain, and (b) leucine oxidation by brain may be influenced by the prevailing glucose and ketone concentrations.     

Permeabilizing effects of sub-inhibitory concentrations of microcystin on the growth of Escherichia coli

Microcystin, a cyanotoxin produced by Microcystis aeruginosa, lacks potent antibacterial activity. When tested in combination, in vitro, inhibitory values for a range of hydrophobic antibiotics were significantly reduced in the presence of at least 1/20 x the minimum inhibitory concentration of microcystin. The degree of inhibition was equivalent to that of a well-characterised permeabilizing agent, polymyxin B nonapeptide. The permeabilizing ability of sub-inhibitory concentrations of microcystin to affect the envelope of Escherichia coli was demonstrated by a rapid and sustained reduction in absorbance values of lysozyme-treated cells and by enhanced uptake of crystal violet in microcystin-treated cultures. Direct effects of appropriate concentrations of microcystin on the integrity of bacterial outer and inner membranes were measured by release of specific enzyme markers. Although the exact mechanism for permeabilizing E. coli with microcystin has not been elucidated, the effects were consistent with permeability changes to the enterobacterial outer membrane caused by polymyxin B nonapeptide.     

Biosynthesis of myo-inositol in rat mammary gland. Isolation and properties of the enzymes

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) and 1l-myo-inositol 1-phosphatase (EC 3.1.3.25) were isolated and partially purified from lactating rat mammary gland. The synthase had an apparent molecular weight of 290,000 as determined by gel filtration; its pH optimum was 7.2, and the K_m for glucose 6-phosphate was 0.5 mm. No other compound could act as a substrate, but the synthase was inhibited 100% by d-gluconic acid 6-phosphate, 54% by d-fructose 6-phosphate, 31.8% by d-galactose 6-phosphate, and 29.6% by d-mannose 6-phosphate each at 5mm. Activity was stimulated 2-fold by the addition of 1 mm NAD⁺ and 40% by 14 mm ammonium ions, whereas it was inhibited by 30% in the presence of 1 mm NADH and by 93.6% when incubated with 1 mmp-mercuribenzoate. Reagents which interfere with Schiff-base formation, pyridoxal 5′-phosphate and trinitrobenzenesulfonate, inhibited the enzyme, but EDTA was without effect.The 1l-myo-inositol 1-phosphatase from rat mammary tissue appears to exist in a native tetrameric form of 210,000 as determined by gel filtration which, upon heating at 70 °C for 15 min, is converted into a stable monomer of approximately 52,000. Mg²⁺ (1.5 mm) was an absolute requirement for activity though Mn²⁺ gave 17% of the activity provided by Mg²⁺. Sodium, potassium, or ammonium ions were stimulatory, but lithium ions were strongly inhibitory. 1l-myo-Inositol 1-phosphatase specifically cleaved 1l-myo-inositol 1-phosphate and was 60% as active toward l-α-glycerol phosphate with only minor activity toward other phosphorylated compounds. The pH optimum was 8.0 and the K_m for 1l-myo-inositol 1-phosphate was 0.8 mm.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

The mode of inhibition of oxidative phosphorylation by efrapeptin (A23871): measurement of substrate effects on rates of inactivation by a tight-binding inhibitor.

Equations are derived for predicting the effects of substrate concentration on the inactivation rate constants of tight-binding competitive and uncompetitive inhibitors. These relationships are used to study the inhibition of mitochondrial oxidative phosphorylation by efrapeptin. The results show that the apparent rate constant for efrapeptin inactivation of ATP synthesis decreases with increase in phosphate concentration. The reciprocal of the observed rate constant varies linearly with changes in the level of phosphate as predicted for a competitive inhibitor. The concentrations of ADP during ATP synthesis and of ATP during ATP hydrolysis, on the other hand, have no effect on the rate of inactivation by efrapeptin. This is in contrast to previous observations that adenine nucleotide substrates influence the level of efrapeptin bound at equilibrium (R. L. Cross, and W. E. Kohlbrenner, 1978, J. Biol. Chem.253, 4865–4873). The results suggest that efrapeptin interacts primarily at the phosphate binding site and that adenine nucleotides may influence equilibrium binding of efrapeptin by affecting the rate of dissociation of the inhibitor. Studies of efrapeptin inhibition of ATP synthesis under pseudo-first-order conditions show that the onset of inhibition is first order with respect to efrapeptin. The maximum apparent rate constant for efrapeptin binding, obtained by extrapolation to zero phosphate concentration, is 1.5 × 10⁵m^?1 s^?1. Also described is a computer program for calculating the concentrations of complexes formed in a mixture of interacting species. The program may be used for most multiple-equilibrium calculations and permits the estimation of the levels of protonated complexes at any pH. The program was used to select Mg²⁺ concentrations which ensure that a large and relatively constant fraction of added ADP is present as MgADP. In the range of phosphate and ADP concentrations commonly used in studies of oxidative phosphorylation a 3 mm excess of Mg²⁺ relative to ADP was found sufficient to maintain high levels of MgADP at pH 8.0.     

The effect of fixed valence metal ions on the free radical process of epinephrine autoxidation

The physiologically active metal ions with fixed valence Ca²⁺ and Mg²⁺ were shown to accelerate epinephrine autoxidation at an alkaline pH, which proceeds via the known quinoid pathway and is accompanied by the generation of reactive oxygen species. A higher efficiency was observed for Ca²⁺ ions compared with Mg²⁺ ions. The activation of epinephrine autoxidation was evident from a decrease in the time of the initiation of the chain reaction to begin (i.e., the reaction lag) and an increase in the rate of both oxygen uptake and the formation of adrenochrome. Based on the observed effects, Ca²⁺ and Mg²⁺ cations were assumed to have the potential to play a role in the free radical processes that are associated with redox reactions in the cell and can also modulate the effect of epinephrine in the organism its oxidation via the quinoid pathway.