A chelating agent possessing cytotoxicity and antimicrobial activity: 7-morpholinomethyl-8-hydroxyquinoline

7-Morpholinomethyl-8-hydroxyquinoline (MO-8HQ), which like 8-hydroxyquinoline (8HQ) readily forms a chelate, was synthesized and found to possess cytotoxicity and antimicrobial activity. Both 8HQ and MO-8HQ were cytotoxic to human carcinoma cell lines at micromolar concentrations. MO-8HQ also inhibited DNA synthesis of tumor cells at micromolar concentrations, suggesting that MO-8HQ might chelate metals necessary for the enzymatic catalysis of DNA biosynthesis. MO-8HQ was more active against gram positive bacteria than gram negative bacteria and its potency correlated with iron chelation. An "unsaturated" chelate with a MO-8HQ to Fe ratio of 2:1 exhibited greater antibacterial activity than MO-8HQ alone. Among the organisms tested, Micrococcus flavus was most susceptible with a MIC of 3.9 microg/ml. MO-8HQ also exhibited anti-fungal activity at 7-15 microg/ml. MO-8HQ:Fe chelate markedly increased the susceptibility of Escherichia coli to deoxycholate. Addition of Ca2+ or Mg2+ reversed the sensitivity of bacteria to deoxycholate as well as to rifampicin. It is suggested that MO-8HQ exerts its biological activity as a membrane-active agent through metal ion chelation.     

Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase.

The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane.     

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.     

Aluminum determination in biological fluids and dialysis concentrates via chelation with 8-hydroxyquinoline and solvent extraction/fluorimetry

We describe a simple, rapid, and sensitive fluorescence method for measurement of aluminum (Al) in human biological fluids, in dialysis solutions, and in tap water, which uses 8-hydroxyquinoline for ion chelation. The fluorescence intensity of the toluene-extracted metal chelate (excitation wavelength, 380 nm; emission wavelength, 504 nm) remains unchanged for over 48 h at room temperature. Fluorescence intensity is a linear function of the concentration of Al in the 2-1000 microg/L range with detection limits of 0.7-2 microg/L. A large excess of other ions normally found in biological fluids does not interfere in Al determination. The method developed was successfully used in assaying Al in serum and urine of reference subjects, in serum samples from patients undergoing long-term dialysis, and in dialysis solutions. Al concentrations, measured by this fluorimetric procedure, were compared with those obtained by Zeeman graphite-furnace atomic absorption spectrometry. A correlation coefficient of 0.98 was obtained. The proposed method could be used for routine analysis in clinical laboratories for accurate determination of aluminum in aqueous or biological fluids.     

Methionyl-tRNA synthetase from Escherichia coli: substituting magnesium by manganese in the L-methionine activating reaction.

While Mg2+ can be efficiently replaced by Ni2+, Co2+ and Mn2+ in the ATP-PPi isotopic exchange reaction catalysed by methionyl-tRNA synthetase from Escherichia coli, the latter ion was selected for detailed analysis of the L-methionine activation reaction. In order to avoid artefactual results due to the slow aggregation of Mn2+ with pyrophosphate, this process was investigated by electron paramagnetic resonance and conditions were determined where it does not interfere with enzymic experiments. The thermodynamic parameters derived from steady-state (ATP-PPi isotopic exchange, fluorescence at equilibrium) or prestationary (fluorescence stopped-flow) experiments are compared to those obtained in the presence of Mg2+ [Hyafil et al. (1976) Biochemistry, 15, 3678-3685]. While the standard deltaG for the reaction (E-Met-ATP-Me2+equilibriumE-Met approximately AMP-PPi-Me2+) is close to zero in the case of Mg2+, Mn2+ slows down the rate of adenylate reversion and thus shifts the reaction towards the latter species. The deltaG for the formation of the E-Met approximately AMP complex does not depend on the metal used, suggesting that the divalent ion does not participate in the structuration of this complex. Substituting Mn2+ for Mg2+ decreases notably the dissociation constant of PPi-Me2+ from the E-Met approximately AMP-PPi-Me2+ species and from its abortive analog E-Met-Ado-PPi-Me2+. Similarly the dissociation constant of ATP-Me2+ from another dead-end analog E-methioninol-ATP-Me2+ is decreased by Mn2+. Involvement of the purine N7 atom in the binding of the metal ion to the active site of methionyl-tRNA synthetase is ruled out by the use of 7-deaza-adenosine. The role of the metal in the catalytic process of methionine activation and its relevance to the specificity of the reaction is then discussed in the light of the results obtained without metal and with Mg2+ and Mn2+.     

Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase.

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.     

Calibration of stopped-flow spectrophotometers using a two-step disulfide exchange reaction

A coupled two-step reaction of Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)) with excess thioglycerol produces a progress curve composed of two superimposed exponentials. The ratio of the two pseudo-first-order rate constants equals 22.5 and does not vary appreciably with ehanges of either pH value or temperature. Because the ratio of the two amplitudes is defined by the ratio of the rate constants, the reaction can be used to estimate both the apparent zero time of mixing (or the dead time) and the detector linearity of a stopped-flow instrument with a single mixing experiment. The reaction is used as a standard of performance for both absorbance and fluorescence measurements with a stopped-flow spectrophotometer.     

Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a Mg2+ ion

Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes. Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site. Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry. Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can be exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative.     

Optical measurement of aqueous potassium concentration by a hydrophobic indicator in lipid vesicles

An assay was developed for K+ in aqueous solution at neutral pH. The method was based on the change in optical absorbance of the hydrophobic indicator 7-(n-decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)indonaphthl++ +-1-ol (MEDPIN) in phospholipid vesicles. Formation of a ternary complex between a valinomycin-K+ pair and the anionic form of MEDPIN in the bilayer resulted in an absorption band at 584 nm. K+ concentration was determined by monitoring the MEDPIN absorbance at 584 nm and MEDPIN quenching of lissamine rhodamine B sulfonylphosphatidylethanolamine (L-RhB-PE) fluorescence by an energy-transfer mechanism. Both the fluorescence intensity and lifetime of L-RhB-PE decreased by more than 25% upon addition of 50 mM K+. Kinetic studies using stopped-flow photometry showed a single-exponential reaction of MEDPIN and valinomycin in vesicles with aqueous K+ (maximum rate 1.7 s-1) that was dependent upon [valinomycin] and [K+]. The lipid surface charge was shown to influence the ratio of anionic to neutral MEDPIN at constant pH, and to alter the sensitivity of MEDPIN absorbance to aqueous [K+]. A 1:20 neutral/negative lipid mole ratio was optimal for K+ detection at pH 7.4. Spectroscopic and kinetic data suggest that the optical response of MEDPIN to K+ involves the formation of a ternary complex between K+, valinomycin and MEDPIN.     

Kinetics of Ca 2+ or Mg 2+ activated ATPase from lymphocyte plasma membranes]

The kinetic study of the C2+ ATPase activity of lymphocyte plasma memebranes allowed some properties of this enzyme to be evidenced. The Ca2+-activated hydrolysis of ATP is independent of a non-specific alkaline phosphatase. The substrate of the ATPase activity is the chelate Ca2+- ATP. Mg2+ may substitute for Ca2+ both as chelating ion and as activating ion. Several results suggest that we have only one ATPase, activated either by Ca2+-, or by Mg2+ with less efficiency; both chelates hve the same Km; pH values for maximum activity and transition temperatures are identical; the effects of free ions are also the same, activation at low concentration and inhibition at high concentration.     

NOTPME: a 31P NMR probe for measurement of divalent cations in biological systems

1,4,7-Triazacyclononane-N,N',N'-tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a 31P NMR indicator of intracellular Mg2+ and Zn2+ ions. The 31P NMR spectrum of this chelate in the presence of metal ions shows characteristic resonances for the free chelate, Mg(NOTPME)-, Zn(NOTPME)-, and Ca(NOTPME)-. The Kd values indicate that this chelate has a 10-fold higher affinity for Mg2+ than for Ca2+ at physiological pH values. In the presence of Mg2+, NOTPME is readily loaded into red blood cells. A 31P NMR spectrum of red cells taken after several washings shows resonances characteristic of entrapped NOTPME and the Mg(NOTPME)- complex, the relative areas of which report an intracellular free Mg2+ concentration of 0.32 mM. The 31P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus-containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentrations and cell energetics simultaneously.     

Plasmid DNA cleavage by MunI restriction enzyme: single-turnover and steady-state kinetic analysis

Mutational analysis has previously indicated that D83 and E98 residues are essential for DNA cleavage activity and presumably chelate a Mg2+ ion at the active site of MunI restriction enzyme. In the absence of metal ions, protonation of an ionizable residue with a pKa > 7.0, most likely one of the active site carboxylates, controls the DNA binding specificity of MunI [Lagunavicius, A., Grazulis, S., Balciunaite, E., Vainius, D., and Siksnys, V. (1997) Biochemistry 36, 11093-11099.]. Thus, competition between H+ and Mg2+ binding at the active site of MunI presumably plays an important role in catalysis/binding. In the present study we have identified elementary steps and intermediates in the reaction pathway of plasmid DNA cleavage by MunI and elucidated the effect of pH and Mg2+ ions on the individual steps of the DNA cleavage reaction. The kinetic analysis indicated that the multiple-turnover rate of plasmid cleavage by MunI is limited by product release throughout the pH range 6.0-9.3. Quenched-flow experiments revealed that open circle DNA is an obligatory intermediate in the reaction pathway. Under optimal reaction conditions, open circle DNA remains bound to the MunI; however it is released into the solution at low [MgCl2]. Rate constants for the phoshodiester bond hydrolysis of the first (k1) and second (k2) strand of plasmid DNA at pH 7.0 and 10 mM MgCl2 more than 100-fold exceed the kcat value which is limited by product dissociation. The analysis of the pH and [Mg2+] dependences of k1 and k2 revealed that both H+ and Mg2+ ions compete for the binding to the same residue at the active site of MunI. Thus, the decreased rate of phosphodiester hydrolysis by MunI at pH < 7.0 may be due to the reduction of affinity for the Mg2+ binding at the active site. Kinetic analysis of DNA cleavage by MunI yielded estimates for the association-dissociation rate constants of enzyme-substrate complex and demonstrated the decreased stability of the MunI-DNA complex at pH values above 8.0.     

Static and kinetic studies on rabbit skeletal muscle troponin

Fluorescence titration curves of 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled troponin (IAANS-labeled Tn) and troponin-1-anilinonaphthalene-8-sulfonic acid (Tn-ANS) complex indicated that the fluorescent moiety, IAANS or ANS, detects conformational change of troponin I (TnI) or Tn due to the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of troponin C (TnC) component. A fluorescence stopped-flow study showed that the kinetic behavior of IAANS-labeled Tn reflects a change in state of the TnI component induced by the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of TnC component. The state change of TnI induced by the Ca2+ binding was complete within the instrumental dead time. On the other hand, that induced by the Ca2+ removal had a rate constant of around 13 s-1. ANS, which is noncovalently bound to Tn, reflects the kinetic properties of both the TnI component and the low affinity Ca2+-binding region of TnC component. The fluorescence intensity change of ANS induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC was complete within the instrumental dead time, while that induced by the Ca2+ removal from the same sites was biphasic. The rate constants of the biphasic process were found to be 62 +/- 7 s-1 and 16 +/- 4 s-1. The former value corresponds to the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TnC component, and the latter value to the rate constant observed in the case of IAANS-labeled Tn. Based on these experimental results and on the discussion in our previous paper (Iio, T. & Kondo, H. (1981) J. Biochem. 90, 163-175), we have refined the two-way information-transfer mechanism which we previously proposed in order to explain the biological function of Tn.     

Troponin-C-mediated calcium-sensitive changes in the conformation of troponin I detected by pyrene excimer fluorescence

Troponin I (TnI) from rabbit white skeletal muscle was labeled at cysteines 48 and 64 with the fluorescent reagent N-(1-pyrene)maleimide. The fluorescence spectra of pyrene-labeled TnI (pyr-TnI) exhibit peaks characteristic of pyrene in its monomeric form and an additional peak resulting from formation of excited dimers (excimers), indicating that the labeled cysteines are close together. Formation of a pyr-TnI-TnC complex in the absence of Ca2+ has little effect on the spectrum, but when Ca2+ is bound to the low-affinity sites of TnC there is a substantial decrease in excimer and a corresponding increase in monomer fluorescence. The involvement of the low-affinity sites in the Ca2+-induced effect is consistent with the fact that Mg2+ has no effect on pyrene fluorescence. On rapid mixing of the pyr-TnI-TnC complex with Ca2+ in a stopped-flow apparatus, most of the excimer decrease is complete within the instrumental dead time, indicating a rate constant k greater than 350 s-1, which is comparable to that of the conformational change in TnC resulting from Ca2+ binding to the low-affinity sites. Rapid mixing of the Mg2-TnC-pyr-TnI complex with Ca2+ yields similar results, suggesting that the type of metal ion present at the high-affinity sites has little, if any, effect on the probe. It has been suggested previously that Cys 48 and 64 are located in a TnT-binding region of TnI (Chong P.C.S. and Hodges, R.S. (1982) J. Biol. Chem. 255, 3757). Our results suggest that a Ca2+-induced structural change in the TnI-binding region of TnC could be transmitted to TnT by affecting the TnT-binding region of TnI as part of the chain of events in the regulation of muscle contraction.     

Ca2+-independent interaction of the gamma subunit of phosphorylase kinase with dansyl-calmodulin

A strong Ca2+-independent interaction between the isolated, active gamma subunit of phosphorylase kinase and dansyl-calmodulin (dansyl-CaM) was observed by monitoring changes in fluorescence intensity in the absence of calcium ion. The pure, active gamma subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive gamma subunit against 8 M urea, containing 0.1 mM DTT, 0.1 M Hepes at pH 6.8 or 0.1 M Tris at pH 8.2, followed by dilution of urea with pH 6.8 or 8.2 buffer. The dissociation constants determined by fluorescence spectroscopy for the gamma subunit to dansyl-CaM are 25.7 +/- 0.6 and 104 +/- 12 nM at pH 6.8 in the presence and absence of CaCl2. At pH 8.2, these values are 4.9 +/- 0.3 and 29 +/- 8 nM in the presence and absence of CaCl2. As the free Ca2+ decreases to as low as 10(-9) M, the fluorescence intensity and the fluorescence polarization of the gamma subunit and dansyl-CaM complex do not decrease in parallel, indicating that the complex does not come apart at low Ca2+ concentration. The presence of Mg2+ affects the interaction between dansyl-CaM and the gamma subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn2+ interferes with the interaction of the gamma subunit and dansyl-CaM. Free ATP has little effect.     

ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation

To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase, we have investigated the effects of ADP, ATP, and analogues of these nucleotides on the rate of dephosphorylation of both native ATPase and ATPase modified with fluorescein 5'-isothiocyanate (FITC), a reagent which hinders access of nucleotides to the ATPase catalytic site without affecting phosphorylation from Pi. Dephosphorylation of the phosphoenzyme formed from Pi was monitored by rapid filtration or stopped-flow fluorescence, mostly at 20 degrees C, pH 6.0, and in the absence of potassium. Fluorescence measurements were made possible through the use of 8-bromo-ATP, which selectively quenched certain tryptophan residues of the ATPase, thereby allowing the intrinsic fluorescence changes associated with dephosphorylation to be measured in the presence of bound nucleotide. ATP, 8-bromo-ATP, and trinitrophenyladenosine diand triphosphate, but not ADP, enhanced the rate of dephosphorylation of native ATPase 2-3-fold when added in the absence of divalent cations. Millimolar concentrations of Mg2+ eliminated the accelerating effects. Acceleration in the absence of Mg2+ was observed at relatively low concentrations of ATP and 8-bromo-ATP (0.01-0.1 mM) and binding of metal-free ATP and ADP, but not Mg.ATP, to the phosphoenzyme in this concentration range was demonstrated directly. Modification of the ATPase with FITC blocked nucleotide binding in the submillimolar concentration range and eliminated the nucleotide-induced acceleration of dephosphorylation. These results show that dephosphorylation, under these conditions, is regulated by ATP but not by Mg.ATP or ADP, and that the catalytic site is the locus of this "regulatory" ATP binding site.     

Mechanism for nucleotide exchange in monomeric actin

Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of inhibition of ribonucleic acid synthesis by 8-hydroxyquinoline and the antibiotic lomofungin.

RNA synthesis in yeast is rapidly inhibited by 8-hydroxyquinoline and the phenazine antibiotic lomofungin (5-formyl-1-methoxycarbonyl-4,6,8-trihydroxyphenazine). It is shown that lomofungin, like 8-hydroxyquinoline, is a chelating agent for bivalent cations. The mechanism of inhibition of RNA synthesis by lomofungin and 8-hydroxyquinoline was investigated in experiments with isolated Escherichia coli RNA polymerase. The results show that both inhibitors are capable of inhibiting polymerase activity solely by chelating the dissociable cations Mn2+ and Mg2+. Evidence is presented which shows that inhibition may occur in the absence of any direct contact between the RNA polymerase or DNA template and the inhibitor. The possibility that inhibition might also occur by chelation of the Zn2+, which is tightly bound to the polymerase, is discussed: it is concluded that lomofungin or 8-hydroxyquinoline is likely to inhibit the enzyme by removal of Mn2+ and Mg2+ before chelating the Zn2+. On the basis of inhibition by chelation of Mn2+ and Mg2+, explanations are proposed for why lomofungin and 8-hydroxyquinoline inhibit synthesis of ribosomal and polydisperse RNA more than that of 5S RNA and tRNA, and for why protein synthesis is not immediately inhibited in the intact yeast cell.     

Two-stage mechanism of the fluoride inhibition of inorganic pyrophosphatase using the fluoride ion

Some kinetic and spectral approaches have been used to study the interactions in the enzyme-Mg2+-F--pyrophosphate (or imidodiphosphate, a non-hydrolyzeable pyrophosphate analog) system underlying the mechanism of yeast inorganic pyrophosphatase inhibition by fluoride. The continuous curves of the enzymatic reaction were obtained with an automatic phosphate analyzer operating on the time scale of seconds. Increasing concentrations of NaF caused an increase in the inactivation rate constant to a constant level of 5.3 min-1 for PPi (pH 6.2-7.2) and 3.9 min-1 for imidodiphosphate, (pH 7.2). At a saturating fluoride concentration, the initial rate of PPi hydrolysis dropped to 10%. NaF and imidodiphosphate changed the protein spectrum at 270-310 nm and strengthened the binding of each other to the protein. The binding of F- required a Mg2+-binding site with Kd = 0.15 mM being filled in. The free enzyme and its Ca2+ complex did not bind F-. The experimental results indicate that pyrophosphatase inhibition by fluoride occurs in two steps. The inhibitor adds first to the Mg2+ ion on the enzyme in a readily reversible reaction causing a 90% decrease of the catalytic activity. Thereafter, a slow isomerization of the enzymesubstrate complex takes place, resulting in a complete loss of activity.     

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.