Inhibition of Water Melon (Citrullus vulgaris) Urease by Fluoride

Effect of fluoride on the activity of purified urease from seeds of watermelon (Citrullus vulgaris) was studied. Fluoride exhibited a concentration dependent inhibition both in presenceand absence ofthe substrate. The inhibition was non-competitive. Addition of 8mM β-mercaptoethanol gradually abolished the fluoride inhibition. β-mnercaptoethanol, in presence of fluoride, also exhibited a concentration dependent suppression of inhibition caused by fluoride. The significance of these observations is discussed.     

Effect of cations on the tyrosine kinase activity of the insulin receptor: Inhibition by fluoride is magnesium dependent

We have recently reported that fluoride interacts directly with the insulin receptor, which causes inhibition of its phosphotransferase activity. The inhibitory effect of fluoride on phosphotransferase activity is not due to the formation of complexes with aluminium and occurs in the absence of alterations to the binding of ATP or insulin. In this report we substantiate that the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle shows a strict requirement of Mg²⁺ ions (Ka near 11 mM). This effect of Mg²⁺ was inhibited in a competitive manner by Mn²⁺, which is compatible with competition of both divalent ions for binding sites. The inhibition of tyrosine kinase activity caused by fluoride was dependent on the concentration of Mg²⁺ in the medium and no inhibitory effect was detected at low concentrations of Mg²⁺. Moreover, the addition of increasing concentrations of Mn²⁺ in the presence of a constant high concentr rease in the inhibitory effect of fluoride. These results indicate that the Mg-insulin receptor complex is the major fluoride-susceptible form. Based on the characteristics of the inhibition of tyrosine kinase shown by fluoride it might be proposed that its action is exerted by the formation of multi-ionic MgF complexes analogous to Pi, which bind to the insulin receptor kinase.     

Modification of the kinetics and regulatory properties of bovine hepatic fructose 1,6-diphosphatase with pyridoxal 5'-phosphate

Treatment of purified fructose 1,6-diphosphatase from bovine liver (which is maximally active at neutral pH) with pyridoxal 5'-phosphate produces changes in the spectral, catalytic, and allosteric properties of the enzyme. After modification the Michaelis constants for fructose-1,6-diphosphate and Mg²⁺ are increased, and inhibition by AMP is decreased. Substrate inhibition is decreased, but not abolished; the curve is merely shifted toward higher substrate concentration. Fructose-1, 6-diphosphate protects against the increases in the K_m for fructose-1, 6-diphosphate and the K_m for Mg²⁺, and against the changes in substrate inhibition, but not against the changes in AMP inhibition. AMP protects against the changes in AMP inhibition and the increase in the K_m for magnesium, but does not prevent the changes in substrate inhibition or the increase in the K_m for fructose-1, 6-diphosphate. The pH curves in the modified enzyme are altered at high, but not at low, substrate concentration.     

Inhibition of Maize Root H+-ATPase by Fluoride and Fluoroaluminate Complexes

Vesicles derived from maize roots retain a membrane-bound H+-ATPase that is able to pump H+ at the expense of ATP hydrolysis. The H+ pumping and the ATPase activity of these vesicles are inhibited by lithium fluoride and by the complex formed between fluoride and aluminum. The inhibition promoted by lithium fluoride increases as the MgCl2 concentration in the medium is increased from 2 to 20 mM. The inhibitory activity of both lithium fluoride and aluminum fluoride increases as the temperature of the medium is increased from 20 to 35[deg]C. Inorganic phosphate (10-40 mM) inhibits the H+ -ATPase at pH 6.5 but not at pH 7.0, and at both pH values, it antagonizes the inhibition promoted by lithium fluoride and fluoroaluminate complexes.     

The adenylate cyclase activity of isolated hepatocytes actions of glucagon and sodium fluoride

Isolated hepatocytes converted exogenous [α-³²P]ATP to cyclic [³²P]AMP at high rates. This system was used for kinetic studies of the effects of glucagon, fluoride, free magnesium and free ATP^4? on adenylate cyclase. In the absence or presence of glucagon, free Mg²⁺ activated adenylate cyclase by decreasing the K_m for MgATP^2? without changing V. Free ATP^4? was not a potent inhibitor of adenylate cyclase and the only effect of glucagon was to increase V.Fluoride also increased the V of adenylate cyclase, but, in contrast to the results obtained with glucagon, the effect increased as the concentration of free Mg²⁺ increased. One explanation of the effect of fluoride, consistent with the idea that free Mg²⁺ activates adenylate cyclase and free ATP is not an inhibitor, is that fluoride increases the affinity of the enzyme for Mg²⁺. Weak inhibition of adenylate cyclase by ATP^4? in the presence of fluoride cannot be excluded.     

Inhibition of co-operative enzymes by substrate-analogues: Possible implications for the physiological significance of negative co-operativity illustrated by phenylalanine metabolism in higher plants

The “Hill” equation for co-operative binding-systems has been extended to describe the effect of substrate-analogue on the binding of substrate to an oligomeric protein. It is demonstrated that the more negatively co-operative the binding-system, the more sensitive is the binding of substrate to inhibition by increases in the relative concentration of substrate-analogue. It is proposed that the physiological significance of negative co-operativity for enzymes may be complementary to the physiological significance of positive co-operativity. The effect of negative co-operativity is to make substrate binding more sensitive to inhibition by relative increases in the concentration of substrate-analogue (e.g. for many enzymes product of the reaction) at the expense of decreased sensitivity of substrate binding to relative changes in substrate concentration compared to a system with equivalent, independent substrate binding sites. In contrast, the effect of positive co-operativity is to make the enzyme more sensitive to relative changes in substrate concentration at the expense of decreased sensitivity to inhibition by relative increases in product concentration, compared to an enzyme without co-operative binding.     

Effect of Fluoride on Nitrification of a Concentrated Industrial Waste

The potential for biological nitrification of an industrial waste containing 4,000 mg of ammonia N (NH₄⁺-N) and 10,000 mg of fluoride per liter was investigated. Ammonium sulfate and sodium fluoride were tested in various combinations of 100 to 2,000 mg of NH₄⁺-N per liter and 0 to 5,000 mg of F⁻ per liter in suspended-growth stirred-tank reactors containing enriched cultures of nitrifying bacteria from a municipal sewage treatment plant. The stirred-tank reactors were fed once per day at a constant hydraulic retention period and cell retention time of 10 days. Temperature was 23°C, and pH was 7.0 to 7.5. Clarified secondary effluent was used to make up feeds and to provide minor nutrients. Steady-state data, confirmed by mass balances, were obtained after five to six retention periods. In the absence of fluoride, nitrification efficiency was near 100% for up to 500 mg of NH₄⁺-N per liter. The influence of fluoride was studied at a low ammonia concentration (100 mg/liter) and exerted no significant effect on nitrification at concentrations of up to 200 mg/liter. Maximum effect of fluoride was reached at 800 mg of F⁻ per liter, and no greater inhibition was observed for up to 5,000 mg of F⁻ per liter. At the highest concentrations studied, ion pairing of ammonium and fluoride may exert a significant effect on kinetic coefficients. Kinetic analyses showed maximum specific substrate removal rates (q_max) of NH₄⁺-N to be about 2.3 mg of N per mg of volatile suspended solids per day in the absence of fluoride and 0.85 mg of N per mg of volatile suspended solids per day in the presence of fluoride. The form of inhibition due to the presence of fluoride was shown to be not competitive, conforming to a mixed inhibition model.     

Fluoride is a slow, tight-binding inhibitor of the calcium ATPase of sarcoplasmic reticulum.

Addition of sodium fluoride in the millimolar concentration range to a solution containing the sarcoplasmic reticulum CaATPase undergoing turnover in its vesicular or nonionic detergent-solubilized forms produced a slow (time range of minutes) complete loss of enzymatic activity. In the presence of magnesium and the absence of calcium, similar results were obtained under nonturnover conditions. Time courses were adequately fit by a function corresponding to a monophasic transformation with a pseudo first order rate constant kobs. In the absence of Mg2+ (EDTA present) no inhibition developed; kobs depended hyperbolically on the Mg2+ concentration with the half maximal effect occurring near 4 mM. The fluoride concentration dependence of kobs showed no evidence of approaching saturation (highest [F-] used was 40 mM) and corresponded to a rate law which was approximately second-order with respect to fluoride. A number of ligands known to bind to the CaATPase were found to decrease kobs. Calcium prevented onset of fluoride inhibition with a midpoint in the micromolar range, implying an effect due to binding at the high affinity transport sites. ATP also protected with a midpoint in the micromolar range, consistent with an effect caused by active site binding of the nucleotide; protection was only partial, suggesting the ATPase can bind fluoride and ATP simultaneously. Prevention of fluoride inhibition by Pi occurred with a [Pi]1/2 of 12 mM at pH 6.5, a concentration similar to that which produces active site phosphorylation. Finally, protection by orthovanadate was found to be competitive and have a midpoint of 5 microM. These results point to an effect exerted at or near the phosphorylation site. The value of kobs increased from essentially zero above pH 8 to a plateau below pH 6; the transition had a midpoint near pH 7.2. Inhibition persisted after removal (with EGTA present) of unbound fluoride by dialysis. Reversal of fluoride inhibition was very slow, with a t1/2 of 16 h at 37 degrees C. These results suggest that fluoride behaves like a slow, tight-binding inhibitor of the ATPase and that the resulting complex is a stable transition (or intermediate) state analog. Plausible molecular bases for our results are that fluoride acts at the phosphorylation site as an analog of Pi or of hydroxide, which may be considered a substrate in the normal hydrolysis of the phosphorylated enzyme. A role for aluminum was ruled out after finding that the addition of EGTA to 10 mM or aluminum sulfate to 0.2 mM or deferoxamine to 0.5 mM produced no significant change in kobs.     

Role of NF-κB transcriptional factor activation during chronic fluoride intoxication in development of oxidative-nitrosative stress in rat’s gastric mucosa

Fluoride compounds are known as hazardous environmental pollutants that can enter the body with drinking water. Chronic exposure to fluoride leads to development of oxidative stress and can lead to activation of nuclear factor κB (NF-κB). The aim of this work is to clarify the role of NF-kB activation in production of reactive nitrogen and oxygen species, activity of antioxidant enzymes and intensity of lipid peroxidation (LPO) in gastric mucosa of rats during chronic fluoride intoxication.Materials and methodsWe carried out the study on 18 mature male rats of the Wistar line. The animals were divided into 3 groups: control animals (6), group of chronic fluoride intoxication (6), and animals (6), which received the NF-κB inhibitor, namely ammonium pyrrolidine dithiocarbamate (PDTC) in a dose of 76 mg / kg (iNF-κB group) during modeling of chronic fluoride intoxication. To assess the development of oxidative stress we studied superoxide production (O₂^-), activity of superoxide dismutase (SOD), catalase (CAT) and concentration of free malondialdehyde (MDA). We also assessed NO production and concentration of its metabolites (peroxynitrite, nitrosilated thiol groups, nitrites).ResultsChronic fluoride intoxication leads to NO hyperproduction with subsequent increase in concentration of its later metabolites (peroxynitrite, nitrosilated thiol groups, nitrites). Production of O₂^- increases, SOD activity decreases, CAT activity increases and MDA concentration also increases. Inhibition of NF-kB activation by PDTC normalizes the parameters studied.ConclusionsActivation of NF-κB during chronic fluoride intoxication leads to the development of hyperproduction of NO and development of oxidative-nitrosative stress.     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

The plasma membrane Ca²⁺?ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeF_x), aluminium fluoride (AlF_x) and magnesium fluoride (MgF_x) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·P_i product states. Our results show that the phosphate analogues BeF_x, AlF_x and MgF_x inhibit PMCA Ca²⁺?ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca²⁺?ATPase inhibition by AlF_x and BeF_x depended on Mg²⁺ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlF_x and BeF_x but not MgF_x apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E?→?E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA.In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Polarity, Protrusion–Retraction Dynamics and Their Interplay during Keratinocyte Cell Migration

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 μg/cm² stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 μg/cm² fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 μg/cm² fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 μg/cm². The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 μg/cm² fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.     

Substrate inhibition of rat liver and kidney arginase with fluoride

Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

NADP-malic enzyme from maize leaf: regulatory properties.

The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4–0.8 mm malate depending on the Mg²⁺ concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 mm Mg²⁺ than at 5–10 mm Mg²⁺ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg²⁺ concentration at 3 mm malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg²⁺ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of $\text{NADPH}\text{NADP}$ increases, and this effect is enhanced at lower pH. (d) Various α-keto acids including glyoxylate, oxaloacetate, and α-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCO₃^?, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO₂ fixation in maize bundle sheath chloroplasts during C₄ photosynthesis.     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.     

Tunicamycin treated fibroblasts secrete a cathepsin B-like protease

We have investigated the effect of tunicamycin on the localization of lysosomal hydrolases in chicken embryo fibroblast cultures. We showed that treatment with tunicamycin (0.05 μg/ml) resulted in a 7–10 fold increase in the cathepsin B-like activity in the culture medium compared to untreated cultures. The protease activity was identified as cathepsin B-like based on 1) substrate specificity (benzoylpro-phe-arg[¹⁴C]anilide is rapidly hydrolyzed), 2) the pH optimum for activity of 5.5, 3) inhibition by thiol reactive compounds, 4) inhibition of the activity by leupeptin but not by pepstatin or phenylmethylsulfonyl fluoride, and 5) by the demonstration of a protease with similar properties in the lysosomal fraction of untreated cultures. The secretion of the cathepsin B-like protease was specific and not due to leakage from damaged cells.