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Originally published In Press as doi:10.1074/jbc.M501563200 on April 29, 2005

J. Biol. Chem., Vol. 280, Issue 50, 41129-41136, December 16, 2005
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Clotrimazole Inhibits Hemoperoxidase of Plasmodium falciparum and Induces Oxidative Stress

PROPOSED ANTIMALARIAL MECHANISM OF CLOTRIMAZOLE*

Vishal Trivedi{ddagger}§1, Prem Chand§, Kumkum Srivastava¶, Sunil K. Puri¶, Prakas R. Maulik{ddagger}, and Uday Bandyopadhyay||2

From the {ddagger}Molecular and Structural Biology Division, Parasitology Division, ||Drug Target Discovery and Development Division, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India and §Department of Physics, Indian Institute of Technology, Kanpur 208016, Uttar Pradesh, India

Received for publication, February 10, 2005 , and in revised form, March 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanism of antimalarial activity of clotrimazole was studied placing emphasis on its role in inhibiting hemoperoxidase for inducing oxidative stress in Plasmodium falciparum. Clotrimazole, in the presence of H2O2, causes irreversible inactivation of the enzyme, and the inactivation follows pseudo-first order kinetics, consistent with a mechanism-based (suicide) mode. The pseudo-first order kinetic constants are ki= 2.85 µM, kinact = 0.9 min-1, and t1/2 = 0.77 min. The one-electron oxidation product of clotrimazole has been identified by EPR spectroscopy as the 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adduct of the nitrogen-centered radical (aN = 15 G), and as DMPO protects against inactivation, this radical is involved in the inactivation process. Binding studies indicate that the clotrimazole oxidation product interacts at the heme moiety, and the heme-clotrimazole adduct has been dissociated from the inactivated enzyme and identified (m/z 1363) by mass analysis. We found that the inhibition of hemoperoxidase increases the accumulation of H2O2 in P. falciparum and causes oxidative stress. Furthermore, the inhibition of hemoperoxidase correlates well with the inhibition of parasite growth. The results described herein indicate that the antimalarial activity of clotrimazole might be due to the inhibition of hemoperoxidase and subsequent development of oxidative stress in P. falciparum.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Clotrimazole (CLT,3 1-({alpha}-2-chlorotrityl)imidazole), an azole derivative, is effective against a wide range of fungal pathogens (1). The fungistatic mechanism of CLT is associated with inhibition of sterol 14 {alpha}-demethylase and microsomal P-450-dependent enzymes (2, 3). Besides antifungal activity, CLT has diverse biological actions. It shows antiproliferative effect by acting as a translation initiation inhibitor (4), inhibits angiogenesis by blocking vascular endothelial growth factor expression (5), decreases glycolysis of lung carcinoma and colon adenocarcinoma cells (6), and induces apoptosis by altering calcium homeostasis of leukemic lymphoblasts (7). It is also known as an effective immunosuppressant (8, 9). CLT inhibits cytochrome P450 monooxygenase and growth of mycobacteria and streptomycetes (10). CLT effectively and rapidly inhibits growth of different strains of Plasmodium falciparum in vitro irrespective of their chloroquine sensitivity (11, 12). The anti-malarial mechanism of CLT is not well understood. It has been suggested that the antimalarial effect may be due to its ability to cause major changes in calcium ion fluxes (13-15). It has been proposed that CLT offers antimalarial effect by inhibiting heme catabolism in the malaria parasite and by enhancing heme-induced membrane damage (16-18). Inhibition of oxidized glutathione (GSSH) export from the infected human red cells or from the parasite itself is also considered to be one possible mechanism of anti-malarial effect of CLT. Efflux of GSSH from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter, and multidrug resistance-associated protein (MRP1) and CLT inhibit the MRP1, which is present in human erythrocytes (19).

The degradation of hemoglobin in the food vacuole of the malaria parasite produces redox-active toxic-free heme, and free heme can produce a significantly high concentration of H2O2 (20-23). H2O2 reduces parasite viability, and the susceptibility of P. falciparum to oxidant-mediated killing has been well established (24-27). Enhanced oxidative stress to the parasite represents a most promising rationale for antimalarial chemotherapy because a number of lines of evidence suggest that this can effectively inhibit parasite growth (28). Peroxidase, a widely distributed enzyme in both the plant and animal kingdom, exerts a wide spectrum of biological functions and plays a very important antioxidant role in defense against oxidative insult of the cell by scavenging the intracellular H2O2 (29-33). We have recently purified and characterized a new heme containing peroxidase (PfHpo) from P. falciparum (34), but its role in the antioxidant defense of the parasite is not yet known. Initial biochemical characterization shows that H2O2 interacts with this hemoperoxidase to form a catalytically active intermediate similar to compound II of peroxidases, where iron remains as a higher oxidation ferryl state (Fe(IV) = 0), and this active intermediate can oxidize aromatic substrate (34). Here we present direct evidence to show that CLT effectively inhibits PfHpo. The mechanism of inhibition has been studied in detail with the purified PfHpo, and the results indicate that CLT inhibits PfHpo by acting as a suicidal substrate. Furthermore, we have shown that the inhibition of PfHpo by CLT is associated with the increased accumulation of H2O2 and oxidative stress in P. falciparum. Moreover, the inhibition of PfHpo correlates well with the inhibition of P. falciparum growth at different concentrations of CLT. The studies, thus, provide new insights on the mechanism of antimalarial activity of clotrimazole.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Saponin, SDS, D-sorbitol, RPMI 1640, clotrimazole, 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), diethylenediaminetriaminopentaacetic acid, dichlorofluorescein diacetate, glutathione (GSH), 5-5'-dithionitrobenzoic acid, thiobarbituric acid, and tetraethoxypropane were purchased from Sigma. Human blood group A+ was drawn from healthy volunteers. H2O2 was purchased from BDH. All other chemicals were of analytical grade purity.

In Vitro Culture of P. falciparum—P. falciparum NF-54 strain was cultured in vitro according to the method of Trager and Jensen (35). P. falciparum was grown in A+ human erythrocytes of 5% hematocrit in RPMI 1640 medium supplemented with 20 mM glucose, 27 mM Na2CO3, pH 7.2-7.4, containing 10% v/v A+ human serum.

Purification and Characterization of PfHpo—PfHpo was purified from P. falciparum as described previously (34). In brief, parasite was isolated from infected red cells, and the isolated parasite was lysed in phosphate-buffered saline by mild sonication (5-s pulse, bath type sonicator) at 4 °C, and the whole lysate was then subcellular-fractionated by differential centrifugation at 500 x g for 10 min, 15,000 x g for 15 min, and 105,000 x g for 1 h to get nuclear, mitochondrial, and microsomal pellet, respectively, and soluble cytoplasmic supernatant. The cytoplasmic supernatant, having peroxidase activity, was subjected to 65% ammonium sulfate precipitation. The supernatant obtained after 65% precipitation was further subjected to 80% ammonium sulfate precipitation, and the enzyme present in the 80% ammonium sulfate cut pellet was finally purified to homogeneity by fast protein liquid chromatography using Superdex-200 HR 10/30 gel filtration column. The purified peroxidase shows a native Mr of ~30 kDa, as calculated from gel filtration by fast protein liquid chromatography using standard molecular weight markers, and it is a dimer of 2 ~15-kDa subunits as evident from SDS-PAGE. The purified peroxidase (R/Z = 2.33, {epsilon}408 = 90 cm-1 mM-1) oxidizes guaiacol in presence of H2O2, and the kcat value is 1.3/s. The Km and Vmax values for guaiacol are 0.7 ± 0.2 mM and 250 ± 40 nmol/min, respectively (34).

Inhibition of PfHpo by CLT—All kinetic measurements were made in PerkinElmer Life Sciences Lambda 15 UV-visible spectrophotometer at 25 ± 1 °C. To measure the peroxidase activity, iodide oxidation was monitored by following the formation of at 353 nm as described previously (31, 36). The rate of inhibition of PfHpo by CLT was measured by incubation of PfHpo (0.5 µM) in the presence of H2O2 (100 µM) and CLT (0.5-5 µM) in a final volume of 30 µl containing 100 mM Tris-HCl buffer, pH 7.2. At various time intervals after the addition of the inhibitor and H2O2, the incubation mixture was transferred to a cuvette containing 1 ml of assay mixture containing 50 mM sodium acetate buffer, pH 5.2, 1.7 mM KI, and 0.27 mM H2O2. During the course of the study of substrate protection against inhibition, electron donor was added to the incubation mixture containing the enzyme before the addition of CLT and H2O2.

EPR Studies—EPR spectra were recorded using DMPO as a spin trap in an X-band EPR spectrometer, Varian E-109, equipped with an E-238 cavity operating in TM 110 mode and using a flat quartz liquid sample cell. PfHpo (10 µM) was incubated with 1 mM H2O2, 1 mM CLT, 1 mM diethylenediaminetriaminopentaacetic acid, and 90 mM DMPO in a total volume of 1 ml containing 100 mM Tris-HCl buffer, pH 7.2. Instrument conditions were: microwave power, 50 milliwatts; microwave frequency, 9.26 GHz; time constant, 0.016 s; modulation frequency, 100 kHz; gain, 2 x 104.

Binding of CLT to PfHpo—Optical spectra were recorded in a total volume of 1 ml containing PfHpo in 100 mM Tris-HCl buffer, pH 7.2, in a PerkinElmer Life Sciences Lambda 15 UV-visible spectrophotometer at 25 ± 1 °C with quartz cells of a 1-cm light path. Binding of CLT to native PfHpo or PfHpo·H2O2 complex was monitored at different concentrations (5-20 µM). A stable PfHpo·H2O2 complex was formed by adding a 10 M excess of H2O2 to the native enzyme, and to this complex different concentrations of CLT were added successively. Soret spectrum was recorded immediately after each addition of CLT.

Heme Dissociation and ESI Mass Analysis—PfHpo (10 µM) was inactivated in the presence of CLT (40 µM) and H2O2 (100 µM) for a period of 10 min at 37 °C in 100 mM Tris-HCl buffer, pH 7.2. Heme was dissociated and extracted from the inactivated enzyme (0.5 ml) by adding an equal volume of cold acetone-HCl mixture (acetone containing 0.015 N HCl) as described (37). Mass of the extracted heme was measured in a Micromass Quattro II triple quadrupole mass spectrometer. The sample was dissolved in methanol and introduced into the ESI source through a syringe pump at the rate of 5 µl/min. The ESI capillary was set at 3.5 kV, and cone voltage was 40 V.

Interaction of H2O2 with Native and CLT-modified PfHpo Measured by Optical Difference Spectroscopy—For modification of PfHpo with CLT, 100 µM H2O2 was added to a microcentrifuge tube containing a solution of excess CLT (1 mM), PfHpo (20 µM), and Tris-HCl buffer (100 mM, pH 7.2) in a total volume of 200 µl and incubated at 37 °C for 30 min. Under these conditions the enzyme showed complete loss of activity. The reaction mixture was passed through the NAP-10 desalting column to remove small molecules. Spectral studies using control and modified enzyme were carried out in a PerkinElmer Life Sciences Lambda 15 UV-visible spectrophotometer at 25 ± 1 °C in a 1-ml cuvette of 1-cm light path. The difference spectra of the CLT-modified PfHpo·H2O2 complex versus CLT-modified PfHpo or native PfHpo·H2O2 complex versus native PfHpo were recorded. Initially, both the sample and reference cuvettes were filled with 1 ml of enzyme solution, and a base-line trace was recorded. A small volume (usually 5-20 µl) of the H2O2 of appropriate concentration was then successively added to the sample cuvette with concomitant addition of the same volume of buffer to the reference cuvette. The contents of each cuvette were stirred well before the difference spectrum was recorded.

Assay of PfHpo (Peroxidase) Activity in P. falciparum—P. falciparum (2% parasitemia) was cultured in the presence or absence of different concentrations of CLT for a period of 48 h. The culture was then washed twice with phosphate-buffered saline, and the parasite was isolated from control and treated groups -as described (38). Isolated parasite was lysed by mild sonication (5-s pulse, bath type sonicator) at 4 °C, and the peroxidase activity was measured from control or inhibitor-treated parasite lysate by following iodide oxidation as described previously (31, 36).

Fluorometric Measurement of Intra-parasitic H2O2—P. falciparum (2% parasitemia) was cultured in the presence or absence of different concentrations of CLT for a period of 48 h. The culture was then further incubated for 30 min in RPMI complete media containing 10 µM 2',7'-dichlorofluorescein diacetate. The culture was then washed twice with phosphate-buffered saline, and the parasite was isolated from control and treated groups as described (38). Isolated parasites were lysed by mild sonication (5-s pulse, bath type sonicator) at 4 °C. H2O2 was measured in control or inhibitor-treated parasite by measuring the fluorescent dichlorofluorescein (DCF) formed due to the oxidation of non-fluorescent probe dichlorofluorescein diacetate by H2O2 as described (39). Fluorescent intensities were recorded from the lysates in a PerkinElmer Life Sciences Lambda LS 50B spectrofluorometer in a 5-mm path length quartz cell in a total volume of 1 ml at wavelength 502 and 523 nm for excitation and emission, respectively. H2O2 was measured as relative fluorescence and expressed as fluorescence intensity/mg of parasite lysate.

Measurement of Reduced Glutathione—P. falciparum (2% parasitemia) was cultured in the presence or absence of different concentrations of CLT. After 48 h of treatment, the culture was washed twice with phosphate-buffered saline, and the parasite was isolated from the infected erythrocytes as described (38). GSH content (as acid-soluble sulfhydryl) from control and inhibitor-treated parasite was determined as described (40, 41). Isolated parasite was sonicated in 100 µl of 20 mM ice-cold EDTA in a bath type sonicator (5-s pulse for 30 s) and centrifuged at 10,000 x g for 20 min to get clear lysate. 50 µl of lysate was mixed with an equal volume of 10% trichloroacetic acid, and protein precipitate was removed by centrifugation. The supernatant was added to an equal volume of 0.8 M Tris-Cl, pH 9, containing 20 mM 5-5'-dithionitrobenzoic acid to yield the yellow chromophore of thionitrobenzoic acid. which was measured at 412 nm. GSH content was measured as nmol/mg of parasite lysate.

Measurement of Lipid Peroxidation as an Index of Oxidative Stress—P. falciparum was cultured in the presence or absence of CLT at different concentrations as described above. Parasite was isolated from infected erythrocytes, and lipid peroxidation products of the parasite lysate were determined as thiobarbituric acid-reactive substances (40, 42). Control or inhibitor-treated parasite (100 µl) was sonicated in ice-cold 0.9% saline in a bath-type sonicator. An aliquot (50 µl) of the parasite lysate was allowed to react with 100 µl of trichloroacetic acid-thiobarbituric acid-HCl reagent containing 0.01% butylated hydroxy-toluene, heated in a boiling water bath for 15 min, cooled, and centrifuged, and the supernatant was used for thiobarbituric acid-reactive substances determination at 535 nm using tetraethoxypropane as standard and expressed as nmol of lipid peroxide/mg of parasite lysate.

Inhibition of Parasite Growth in Culture—P. falciparum culture was synchronized by D-sorbitol treatment to obtain rings (43), and all assays were started with ring-synchronized cultures of 2-4% parasitemia and processed as described previously (12). Aliquots of stock solutions of CLT in Me2SO were dispensed in cell culture plates under sterile conditions to make final concentrations of 0.1-2 µM after the addition of parasitized red cell suspension in culture medium. The plates were placed in gas-tight desiccators that were flushed with a low oxygen gas mixture, sealed, and incubated at 37 °C for 0-48 h. Parasitemia and differential counts of asexual stages were carried out by counting parasites per 5000 erythrocytes after Giemsa staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of PfHpo by CLT—Preincubation of PfHpo with increasing concentrations of CLT in the presence of fixed H2O2 concentration results in concentration- and time-dependent irreversible inactivation of the enzyme after pseudo-first order kinetics (Fig. 1A), consistent with a mechanism-based (suicide) mode. When Kobs obtained from the slope of each line was plotted against CLT concentration, a straight line (Fig. 1B) was obtained from which a second-order rate constant was calculated to be 10 x 104 M-1·min-1 at 25 °C. The half-life of the enzyme inactivation (t1/2) at each CLT concentration, when plotted against the reciprocal of CLT concentration, yields a straight line (Fig. 1C). From this plot the affinity and kinetic constants were calculated, and the values of ki, kinact, and t1/2 were found to be 2.85 µM, 0.9 min-1, and 0.77 min, respectively. No significant inactivation occurs in the absence of H2O2 or CLT, suggesting the involvement of PfHpo-catalyzed CLT (CLTox) in the inactivation process. The results indicate that only H2O2 or CLT fails to inhibit PfHpo significantly, whereas the activity is almost completely inhibited when PfHpo is incubated in the presence of both CLT and H2O2 (TABLE ONE). Moreover, no catalytic activity could be recovered by either dilution of the reaction mixture or removing CLT and H2O2 by NAP 10 desalting column chromatography (TABLE ONE), indicating irreversible inactivation of the enzyme. Thus, CLT acts as a suicidal substrate and inactivates PfHpo after mechanism-based kinetics.


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  		TABLE ONE
Suicidal inhibition of PfHpo by CLT PfHpo (0.5 µM) was incubated in the presence or absence of CLT (2 µM) or H2O2 (100 µM) for 5 min at 37 °C in a total volume of 30 µl containing 100 mM Tris-HCl buffer, pH 7.2. The reaction mixture containing the CLT-inactivated enzyme was passed through NAP-10 desalting column, and the flow-through was collected for activity assay. Peroxidase activity was measured by following iodide oxidation as described under "Experimental Procedures."

 



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  		Fig. 1.
Kinetics of the inactivation of PfHpo by CLT in the presence of 100µM H2O2. A, calculation of the pseudo-first order rate constant of inactivation for iodide oxidation. PfHpo (0.5 µM) was incubated with different concentrations of CLT in the presence of 100 µM H2O2 at 25 ± 1 °C in a final volume of 1 ml containing 100 mM Tris-HCl buffer, pH 7.2. The data were plotted as described under "Results." The concentrations of CLT used are indicated in the parentheses. B, determination of the second-order rate constant of inactivation of iodide oxidation by PfHpo. The slopes of the straight lines obtained in A were plotted against the concentration of CLT. The slope of this line indicates the second-order rate constant of inactivation, which is 10 x 104·M-1·min-1. C, kinetics of the mechanism-based inactivation of PfHpo by CLT. The times required for the half-time of inactivation at each concentration of CLT obtained from the straight lines of A were plotted against their corresponding reciprocal CLT concentrations. Kinetic constants (t1/2) were calculated from the y axis intercept, and ki was from the x axis intercept of the straight line. kinact was calculated by dividing the first order rate constant (0.69) by the t1/2 value (57). Data are the means ± S.E. (n = 3).

 
Evidence for One-electron Oxidation of CLT—Peroxidase plays a very important role in host defense against oxidative damage by scavenging the intracellular H2O2 through the oxidation of a large variety of substrates including aromatic donors and inorganic anions (29-33). Because CLT acts as a suicidal substrate to PfHpo, it must be oxidized by the catalytically active PfHpo before inactivation. EPR spectroscopy using spin trap DMPO indicates that PfHpo oxidizes CLT by one-electron oxidation, and the formation of the CLT free radical has been identified as DMPO-CLT radical adduct (aN = 15 G) in the PfHpo·H2O2 system (Fig. 2a). This radical is not formed in the absence of H2O2 (Fig. 2b) or CLT (Fig. 2c).



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  		Fig. 2.
EPR spectra of the DMPO-CLT radical adduct. a, EPR spectrum obtained with 10 µM PfHpo, 1 mM CLT, 1 mM H2O2, 90 mM DMPO, and 1 mM diethylenediaminetriaminopentaacetic acid in 100 mM Tris-HCl buffer, pH 7.2. b,asfor a but in the absence of H2O2; c, as for a but in the absence of CLT.

 
Protection of PfHpo against CLT Inactivation by Electron Donors— The kinetic data presented so far indicate that PfHpo is inactivated irreversibly by CLT, which acts as a suicidal substrate for oxidized PfHpo. However, inactivation could be prevented if the enzyme was preincubated with CLT in the presence of another electron donor (substrate) such as iodide, bromide, chloride, and guaiacol. TABLE TWO shows the effect of electron donors on PfHpo inactivation. All the electron donors quite effectively protect the enzyme from inactivation, but iodide or chloride protects in a better degree when compared with bromide or the aromatic donor guaiacol at the same concentration (100 µM). CLT alone could inhibit 98% activity, but in the presence of 100 µM iodide or chloride, it could inhibit only 5-6%.


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  		TABLE TWO
Protection of PfHpo against CLT inactivation by electron donors PfHpo (0.5 µM) was incubated in the presence or absence of electron donors (100 µM) in a total volume of 30 µl containing 100 mM Tris-HCl buffer, pH 7.2, CLT (2 µM), and H2O2 (100 µM) for 5 min at 37 °C. Peroxidase activity of the incubation mixture was measured by following iodide oxidation as described under "Experimental Procedures."

 
Demonstration That CLT Oxidation Product Is the Inactivating and Interacting Species—The results in Fig. 3 indicate that, whereas PfHpo is inactivated on preincubation with CLT and H2O2, it is almost completely protected in the presence of the spin trap DMPO. The protection against inhibition of PfHpo by CLT by DMPO indicates that the one-electron oxidation product of CLT is the inactivating species. To find out whether CLT or its oxidation species interacts with the active enzyme, spectral studies were carried out with the native and catalytic complex (PfHpo·H2O2 complex). CLT at a concentration of 5-10 µM interacted at the heme site of the catalytically active enzyme (PfHpo·H2O2 complex) as evidenced from the decrease of absorbance of in Soret peak at 413 nm (Fig. 4A), whereas we observed virtually no binding of CLT with the native enzyme even at a concentration of 20 µM (Fig. 4B), indicating that oxidation product is the possible interacting species. To see whether the binding of CLT oxidation product is reversible or irreversible, we extracted heme from the enzyme after CLT inactivation and analyzed its mass by ESI mass spectroscopy. Appearance of a product having an m/z of 1363 {heme + 2x (clotrimazole) + Na + Cl} (Fig. 5A) clearly indicates the formation of heme-CLT adduct and irreversible binding of CLT oxidation product to the heme moiety of the enzyme. Moreover, when PfHpo is incubated in the absence of clotrimazole (Fig. 5B) or H2O2 (Fig. 5C), the extracted heme only shows the prominent peak of heme (m/z 616), and the heme-clotrimazole adduct (m/z 1363) is not formed, indicating the requirement of both CLT and H2O2 for suicidal inactivation. Besides the peak of the heme-CLT adduct (m/z 1363), there are many prominent peaks (e.g. m/z 277, 616, 681, 779, 900, 998, 1119, and 1262) in the spectrum of heme extracted from CLT-inactivated PfHpo (Fig. 5A). These peaks are originated due to the fragmentation of the adduct, and the probable nature of these peaks are as follows; m/z 277 {clotrimazole - imidazole (C3H3N2)}, m/z 616 {Heme}, m/z 681 {heme + imidazole (C3H3N2)}, m/z 779 {heme + imidazole (C3H3N2) + C7H5 + Li}, m/z 998 {heme + imidazole (C3H3N2) + CLT + Li - Cl}, m/z 900 {heme + imidazole (C3H3N2) + clotrimazole - C7H5 - Cl}, m/z 1119 {M+ (1363) - 2 x imidazole (C3H3N2) - C6H5 - Cl} and m/z 1262 {M+ (1363) - imidazole (C3H3N2) - Cl}.



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  		Fig. 3.
Protection against CLT inhibition by spin trap DMPO. PfHpo (0.5 µM) was preincubated in the presence or absence of DMPO at different concentrations as indicated in presence of CLT (5 µM) and H2O2 (100 µM) in a total volume of 30 µl containing 100 mM Tris-HCl buffer, pH 7.2. The incubation mixture was transferred to a cuvette containing 1 ml of assay mixture containing 50 mM sodium acetate buffer, pH 5.2, 1.7 mM KI, and 0.27 mM H2O2, and peroxidase activity was assayed as described under "Experimental Procedures."

 



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  		Fig. 4.
Binding of CLT to catalytically active (PfHpo·H2O2 complex) and native PfHpo. The spectra were taken in a total volume of 1 ml of 100 mM Tris-HCl buffer, pH 7.2. Panel A: a, native PfHpo (2.5 µM); b, stable PfHpo·H2O2 complex formed after the addition of 10 eq of H2O2 (25 µM) to PfHpo; c, b + CLT (5 µM); d, c + CLT (10 µM); e, d + CLT (20 µM). B: a, native PfHpo (4.5 µM); b, a + CLT (5 µM); c, b + CLT (10 µM); d, c + CLT (20 µM).

 
CLT-inactivated Enzyme Can Form a Complex by Interacting with H2O2—H2O2 interacts with the heme iron of peroxidases to form an active enzyme intermediate like compound I or compound II, and the formation of these enzyme intermediates is essential for substrate oxidation (29, 33). We have shown that CLT-inactivated enzyme fails tooxidize peroxidase substrate like iodide. To know whether this is due to the absence of H2O2 interaction with the inactivated enzyme to form active enzyme intermediate, PfHpo was inactivated with H2O2 and CLT, and spectral studies were carried out to detect the enzyme intermediate (PfHpo·H2O2 complex) formation after removal of the small molecules through a spin column of G-25. Fig. 6 shows that CLT-inactivated enzyme can readily form a complex with H2O2. Interaction of H2O2 with the native enzyme (Fig. 6A) or CLT-inactivated enzyme (Fig. 6B) is more or less the same at different concentrations of H2O2 (25-80 µM). This indicates that heme iron remains free in the inactivated enzyme to interact with H2O2, or the interaction of the CLT oxidation product does not interfere with the reactivity of the enzyme with H2O2.



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  		Fig. 5.
Identification of heme-CLT adduct by ESI-mass spectroscopy. A, ESI mass spectroscopy spectrum of the heme extracted from the inactivated PfHpo (10 µM) in presence of CLT (40 µM) and H2O2 (100 µM). B, as for A but in the absence of CLT. C, as for A but in the absence of H2O2.

 



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  		Fig. 6.
Interaction of H2O2 with native and CLT-inactivated PfHpo. A, optical difference spectra of native PfHpo·H2O2 complex formation at different concentrations of H2O2 (25, 50, 70, 80, and 100 µM)(i, initial scan at 25 µM; f, final scan at 100 µM). B, optical difference spectra of inactivated PfHpo·H2O2 complex formation at different concentrations of H2O2 (25, 50, 70, and 80 µM) (i, initial scan at 25 µM; f, final scan at 80 µM)

 
CLT Inhibits PfHpo and Induces Oxidative Stress in P. falciparum—To see the effect of PfHpo inhibition by CLT on parasite growth, P. falciparum was cultured in the presence or absence of different concentrations of CLT. CLT inhibits PfHpo of P. falciparum in a concentration-dependent manner with 50% inhibition at 0.5 µM and 90% inhibition at 2 µM (Fig. 7A), and the inhibition of PfHpo is associated with the significant accumulation of H2O2 in parasite (Fig. 7B). CLT increases 50 and 100% H2O2 accumulation (as compared with control) in parasite at a concentration of 0.6 and 0.8 µM, respectively (Fig. 7C). The generation of intra-parasitic H2O2 and the inhibition of PfHpo are well correlated at different concentrations of CLT (Fig. 7C), indicating that the inhibition of PfHpo may be the cause for higher H2O2 generation. Moreover, the inhibition of PfHpo is well correlated with the inhibition of parasite growth (Fig. 7D). 90% inhibition of PfHpo at 2 µM CLT causes almost 100% inhibition of parasite growth. To further confirm the development of oxidative stress due to the inhibition of PfHpo, GSH level and lipid peroxidation products are measured from control and CLT-treated parasite. CLT concentration dependently increases lipid peroxidation and decreases GSH level (TABLE THREE). CLT at a concentration of 0.5 µM increases 50% lipid peroxidation and decreases 36% GSH, but at a concentration of 2 µM it increases 100% lipid peroxidation and decreases 64% GSH level in P. falciparum (TABLE THREE).


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  		TABLE THREE
Effect of CLT on lipid peroxidation and GSH content in P. falciparum GSH and lipid peroxidation products were measured from the control and inhibitor-treated parasites as described under "Experimental Procedures." The values presented were the mean ± S.E. of three different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Evidence has been presented to show that CLT inhibits P. falciparum hemoperoxidase, and the inhibition is associated with the development oxidative stress and inhibition of P. falciparum growth. CLT is also known to inhibit heme-containing enzymes (44, 45). Another antifungal azole, miconazole, inhibits peroxidase, and the possible involvement of peroxidase in the mechanism of action of this drug has been suggested (46). Kinetic evidence shows that CLT inactivates PfHpo by acting as a suicidal substrate. Peroxidases oxidize varieties of aromatic substrates through one-electron oxidation in the presence of H2O2 (29, 33). Evidence has been presented to show that CLT is oxidized to a highly reactive free radical before irreversibly inactivating PfHpo. Peroxidase in normal ferric state reacts with H2O2 to form a transient green complex, compound I, which undergoes a one-electron reduction by an electron donor to form relatively stable compound II (29, 33, 47). The latter further undergoes a one-electron reduction by a second electron donor to regenerate ferriperoxidase to start a new cycle as,

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)
where AH2 represents the aromatic electron donor, and AH. is the corresponding one-electron oxidation product (free radical). The radicals may dimerize to generate stable oxidation product A. But in the case of the oxidation of suicidal substrates by peroxidases, substrate free radicals formed interact with the enzyme to inactivate it rather forming a stable polymer of oxidation product (36, 48-50). When CLT was added to PfHpo in the presence of H2O2, CLT was oxidized by a one-electron transfer process, probably by the ferryl state of the enzyme, to form N-centered CLT radicals, which were conveniently detected by EPR spectroscopy as the CLT radical-DMPO adduct, having a splitting constant aN = 15 G. Interestingly, DMPO, the spin trap, can protect enzyme from inactivation because CLT free radicals are no longer available. This further proves that CLT free radicals act as essential intermediates to inactivate the enzyme. EPR studies were done in the presence of diethylenediaminetriaminopentaacetic acid (1 mM), a known metal chelator; therefore, we exclude the possibility that the observed spectrum does not arise from the reaction of trace metal ions perhaps released from the peroxidase. We also exclude the possible involvement of non-enzymatically formed hydroxyl radical or superoxide radical in the process of inactivation, as EPR parameters are not consistent with the splitting constants of hydroxyl or superoxide radicals (51, 52). Inactivation of enzyme by suicidal substrate must be protected by its putative substrates. Putative peroxidase substrates of peroxidase such as iodide, bromide, and chloride protect PfHpo against inactivation. Aromatic substrate, guaiacol, is also capable of protecting the enzyme. Optical spectroscopic studies indicate that CLT binds at the heme moiety of oxidized enzyme (PfHpo·H2O2 complex) but not to the native enzyme, further indicating that the oxidation product of CLT is the interacting species. Suicidal substrates of peroxidase are known to inactivate via generation of a more reactive radical that interacts with the specific heme site to block electron transfer (53-56). Evidence has been presented to show that CLT binds irreversibly at the heme moiety of this peroxidase, and heme-CLT adduct has been dissociated from the inactivated enzyme. The heme-CLT adduct shows a molecular ion peak at m/z 1363 {heme + 2x (clotrimazole) + Na + Cl} in ESI mass spectroscopy. Analysis of the mass spectrum of heme-CLT adduct indicates that two molecules of CLT probably interact with one molecule of heme. Evidence has been presented to show that inactivated enzyme can still form a complex with H2O2-like native enzyme, but it fails to oxidize peroxidase substrate (iodide). CLT oxidation product probably attacks a specific heme site that may be close or at substrate binding site, or binding of CLT prevents a transfer of the electron from iodide to the heme ferryl group. However, further studies are necessary to confirm the above hypothesis.



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  		Fig. 7.
Inhibition of PfHpo is associated with the accumulation of intra-parasitic H2O2 and inhibition of P. falciparum growth. Inhibition of PfHpo activity (A) and generation of H2O2 (B) at different concentrations of CLT as indicated. C, correlation of PfHpo inhibition with the formation of H2O2 at different concentrations of CLT. D, correlation of inhibition of PfHpo with the inhibition of parasite growth. Parasites were exposed for 48 h to serial dilutions of inhibitor. Values were presented as the mean number of parasites per 103 red blood cells. Detailed methodology was described under "Experimental Procedures."

 
We found that the inhibition of PfHpo by CLT is associated with the increased accumulation of intra-parasitic H2O2 and enhanced oxidative stress. Moreover, inhibition of PfHpo correlates well with the inhibition of P. falciparum growth in vitro. The malarial parasite is susceptible to oxidative stress (24, 26, 27). Peroxidase plays a significant antioxidant role in defense against the oxidative insult of the cell by scavenging the intracellular H2O2 (29-33). Therefore, the inhibition of PfHpo by CLT leads to accumulation of intraparasitic H2O2, which should cause lipid peroxidation or reduced GSH levels. Evidence has been presented to show that PfHpo inhibition is associated with the formation of lipid peroxidation product and the fall of GSH in the parasite. The inhibition of PfHpo is correlated well with the inhibition of P. falciparum growth at different concentrations of CLT, indicating the possible role of oxidative stress developed due to the inhibition of PfHpo to inhibit parasite growth. Earlier it was shown that CLT had time- and concentration-dependent effects with a broad stage specificity of action, and it was more effective at the late trophozoites and schizont stages than at the ring-trophozoite stage (12). The ring stage of parasite is comparatively less metabolically active in comparison to the late trophozoite and schizont stages. The late trophozoites and schizont stages, due to a higher metabolic rate, cause degradation of very high amounts of hemoglobin releasing redox-active free heme. A higher metabolic rate and free heme may cause oxidative stress due to the generation of reactive oxygen species, particularly H2O2 (20-23). Therefore, the probability of the generation of reactive oxygen species and subsequent oxidative stress is less in the ring and early trophozoite stages. PfHpo, by scavenging intracellular H2O2, probably protects the parasite against this oxidative stress. CLT is more effective at the late trophozoites and schizont stages as it causes enhanced oxidative stress by inhibiting PfHpo. We now suggest from the present study that CLT induces oxidative stress in P. falciparum probably by irreversibly inhibiting PfHpo, and enhancement of oxidative stress by inhibiting PfHpo is a novel antimalarial effect of clotrimazole.

Note Added in Proof—A Blast search of N-terminal sequence VLSPADKTNV in the protein data base SWISSPROT and circular dichroism analysis of the heme-containing peroxidase purified from P. falciparum revealed that it is identical to hemoglobin (human) {alpha} chain. Moreover, immunoblot analysis of the protein by using both antibodies generated against this protein and human hemoglobin further revealed that this protein originated from host hemoglobin. As the protein has N-terminal sequence homology with the {alpha} chain, not with the {beta} chain (VHLTPEEKSA), with native and subunit molecular masses of ~30 and 15.118 kDa, respectively, it is likely a dimer of {alpha} chain. However, more careful analysis of subunit mass indicates that it has an m/z value of 15,118, which is 609 mass units lower than that of intact heme-containing {alpha} chain (m/z 15,727). Since the N-terminal sequence is intact, the data suggest that this protein is a C-terminal modified (deletion of few C-terminal amino acids) dimer of {alpha} chain (molecular mass of the intact heme-containing {alpha} dimer is ~31.45 kDa). Alternatively, the experimental m/z value of the subunit may also correspond to the molecular ion of the {alpha} chain with the loss of the heme moiety. However, in P. falciparum, generation of {alpha} and {beta} chains truncated at their C terminus after hemoglobin breakdown has already been reported (20). However extensive studies are necessary to find out any specific protease or actual process responsible for the formation of this {alpha} dimer in P. falciparum.


   FOOTNOTES
 
* This work was supported in part by the Council of Scientific and Industrial Research (CSIR), New Delhi, for providing grants through the CSIR Networked Project (SMM03 (P22)). This is CDR1 Communication No. 6771. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a Council of Scientific and Industrial Research fellowship. Back

2 To whom correspondence should be addressed. Tel.: 91-522-2612411; Fax: 91-522-2613405; E-mail: ubandyo_1964{at}yahoo.com.

3 The abbreviations used are: CLT, clotrimazole; DMPO, 5,5'-dimethyl-1-pyrroline N-oxide; ESI, electrospray ionization; PfHpo, P. falciparum hemoperoxidase. Back


   ACKNOWLEDGMENTS
 
We acknowledge the Sophisticated Analytical Instrumentation Facility, Central Drug Research Institute, Lucknow for facilitating of all spectroscopic studies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sawyer, P. R., Brogden, R. M., and Pinder, R. M. (1975) Drugs 9, 424-447[Medline] [Order article via Infotrieve]
  2. Bossche, H. V., Willemsens, G., Cools, W., Marichal, P., and Lauwers, W. (1983) Biochem. Soc. Trans. 11, 665-667[Medline] [Order article via Infotrieve]
  3. Yoshida, Y., and Aoyama, Y. (1987) Biochem. Pharmacol. 36, 229-235[CrossRef][Medline] [Order article via Infotrieve]
  4. Natarajan, A., Fan, Y. H., Chen, H., Guo, Y., Iyasere, J., Harbinski, F., Christ, W. J., Aktas, H., and Halperin, J. A. (2004) J. Med. Chem. 47, 1882-1885[CrossRef][Medline] [Order article via Infotrieve]
  5. Takei, S., Iseda, T., and Yokoyama, M. (2003) Int. J. Urol. 10, 78-85[CrossRef][Medline] [Order article via Infotrieve]
  6. Penso, J., and Beitner, R. (2002) Eur. J. Pharmacol. 45, 227-235
  7. Ito, C., Tecchio, C., Coustan-Smith, E., Suzuki, T., Behm, F. G., Raimondi, S. C., Pui, C. H., and Campana, D. (2002) Leukemia 16, 1344-1352[CrossRef][Medline] [Order article via Infotrieve]
  8. Khanna, R., Chang, M. C., Joiner, W. J., Kaczmarek, L. K., and Schlichter, L. C. (1999) J. Biol. Chem. 274, 14838-14849[Abstract/Free Full Text]
  9. Jensen, B. S., Odum, N., Jorgensen, N. K., Christophersen, P., and Olesen, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10917-10921[Abstract/Free Full Text]
  10. McLean, K. J., Marshall, K. R., Richmond, A., Hunter, I. S., Fowler, K., Kieser, T., Gurcha, S. S., Besra, G. S., and Munro, A. W. (2002) Microbiology 148, 2937-2949[Abstract/Free Full Text]
  11. Saliba, K. J., and Kirk, K. (1998) Trans. R. Soc. Trop. Med. Hyg. 92, 666-667[CrossRef][Medline] [Order article via Infotrieve]
  12. Tiffert, T., Ginsburg, H., Krugliak, M., Elford, C., and Lew, V. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 331-336[Abstract/Free Full Text]
  13. Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nat. Med. 1, 534-540[CrossRef][Medline] [Order article via Infotrieve]
  14. Tiffert, T., Staines, H. M., Ellory, J. C., and Lew, V. L. (2000) J. Physiol. (Lond.) 525, 125-134[Abstract/Free Full Text]
  15. Aktas, H., Fluckiger, R., Acosta, J. A., Salvage, J. M., Palakurthi, S. S., and Halperin, J. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8280-8285[Abstract/Free Full Text]
  16. Huy, N. T., Kamei, K., Yamamoto, T., Kondo, Y., Kanaori, K., Takano, R, Tajima, K., and Hara, S. (2002) J. Biol. Chem. 277, 4152-4158[Abstract/Free Full Text]
  17. Huy, N. T., Kamei, K., Kondo, Y., Serada, S., Kanaori, K., Takano, R, Tajima, K., and Hara, S. (2002) J. Biochem. (Tokyo) 131, 437-444[Abstract/Free Full Text]
  18. Huy, N. T., Takano, R., Hara, S., and Kamei, K. (2004) Biol. Pharm. Bull. 27, 361-365[CrossRef][Medline] [Order article via Infotrieve]
  19. Klokouzas, A., Barrand, M. A., and Hladky, S. B. (2001) Eur. J. Biochem. 268, 6569-6577[Medline] [Order article via Infotrieve]
  20. Rosenthal, P. J., and Meshnick, S. R. (1998) Malaria: Parasite Biology, Pathogenesis and Protection (Sherman, I. W., ed) pp. 145-158, American Society for Microbiology, Washington, D. C.
  21. Francis, S. E., Gluzman, I. Y., Oksman, A., Knickerbocker, A., Mueller, R., Bryant, M. L., Sherman, D. R., Russell, D. G., and Goldberg, D. E. (1994) EMBO J. 13, 306-317[Medline] [Order article via Infotrieve]
  22. Foley, M., and Tilley, L. (1998) Pharmacol. Ther. 79, 55-87[CrossRef][Medline] [Order article via Infotrieve]
  23. Wright, A. D., Wang, H., Gurrath, M., Konig, G. M., Kocak, G., Neumann, G., Loria, P., Foley, M., and Tilley, L. (2001) J. Med. Chem. 44, 873-885[CrossRef][Medline] [Order article via Infotrieve]
  24. Wozencraft, A. O., Dockrell, H. M., Taverne, J., Target, G. A. T., and Playfair, J. H. L. (1984) Infect. Immun. 43, 664-669[Abstract/Free Full Text]
  25. Ockenhouse, C. F., Schulman, S., and Shear, H. L. (1984) J. Immunol. 133, 1601-1608[Abstract]
  26. Friedman, M. J. (1979) Nature 280, 245-247[CrossRef][Medline] [Order article via Infotrieve]
  27. Clark, I. A. N., and Hunt, H. (1983) Infect. Immun. 39, 1-6[Abstract/Free Full Text]
  28. Becker, K., Tilley, L., Vennerstrom, J. L., Roberts, D., Rogerson, S., and Ginsburg, H. (2004) Int. J. Parasitol. 34, 163-189[CrossRef][Medline] [Order article via Infotrieve]
  29. Dunford, H. B., and Stillman, J. S. (1976) Coord. Chem. Rev. 19, 187-251[CrossRef]
  30. Pruitt, K. M., and Tenovuo, J. O. (1985) The Lactoperoxidase System: Chemistry and Biological Significance, Marcel Dekker, Inc., New York
  31. Bandyopadhyay, U., Bhattacharyya, D. K., Chatterjee, R., and Banerjee, R. K. (1992) Biochem. J. 284, 305-312[Medline] [Order article via Infotrieve]
  32. Bandyopadhyay, U., Chatterjee, R., Chakraborty, T. K., Ganguly, C. K., Bhattacharyya, D. K., and Banerjee, R. K. (1997) Biochem. Pharmacol. 54, 241-248[CrossRef][Medline] [Order article via Infotrieve]
  33. Dunford, H. B. (1991) Peroxidase in Chemistry and Biology (Everse, S., Everse, K. E., and Grisham, M. B., eds) pp. 1-24, CRC Press, Inc., Boca Raton, FL
  34. Trivedi, V., Srivastava, K., Puri, S. K., Maulik, P. R., and Bandyopadhyay, U. (2005) Protein Expression Purif. 41, 154-161[CrossRef][Medline] [Order article via Infotrieve]
  35. Trager, W., and Jensen, J. B. (1976) Science 193, 673-675[Abstract/Free Full Text]
  36. Bandyopadhyay, U., Bhattacharyya, D. Kr., Chatterjee, R., and Banerjee, R. K. (1995) Biochem. J. 306, 751-757[Medline] [Order article via Infotrieve]
  37. Murphy M. J., Siegel L. M., Tove, S. R., and Kamin, H. (1974) Proc. Nat. Acad. Sci. U. S. A. 71, 612-616[Abstract/Free Full Text]
  38. Hsiao, L.L., Howard, R. J., Aikawa, M., and Taraschi, T. F. (1991) Biochem. J. 274, 121-132[Medline] [Order article via Infotrieve]
  39. Munzel, T., Afanas'ev, I., Kleschyov, A., and Harrison, D. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 1761-1768[Abstract/Free Full Text]
  40. Bandyopadhyay, U., Biswas, K., Chatterjee, R., Bandyopadhyay, D., Chattopadhyay, I., Ganguly, C. K., Chakraborty, T., Bhattacharyya, K., and Banerjee, R. K. (2002) Life Sci. 71, 2845-2865[CrossRef][Medline] [Order article via Infotrieve]
  41. Das, D., Bandyopadhyay, D., Bhattacharyya, M., and Banerjee, R. K. (1997) Free Radic. Biol. Med. 23, 8-18[CrossRef][Medline] [Order article via Infotrieve]
  42. Biswas, K., Bandyopadhyay, U., Chattopadhyay, I., Varadaraj, A., Ali, E., and Banerjee, R. K. (2003) J. Biol. Chem. 278, 10993-11001[Abstract/Free Full Text]
  43. Lambros, C., and Venderberg, J. P. (1979) J. Parasitol. 65, 418-420[CrossRef][Medline] [Order article via Infotrieve]
  44. Tiran, V. K., Mishin, V. M., and Thomas, P. E. (2001) Drug Metab. Dispos. 29, 837-842[Abstract/Free Full Text]
  45. Sennequier, N., Wolan, D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 930-938[Abstract/Free Full Text]
  46. Nollin, S. D., Belle, H. V., Goossens, F., Thone, F., and Borgers, M. (1977) Antimicrob. Agent. Chemother. 11, 500-513[Abstract/Free Full Text]
  47. Chance, B. (1952) Arch. Biochem. Biophys. 41, 404-415[CrossRef][Medline] [Order article via Infotrieve]
  48. Doerge, D. R. (1986) Biochemistry 25, 4724-4728[CrossRef][Medline] [Order article via Infotrieve]
  49. Engler, H., Taurog, A., and Nakashima, T. (1982) Biochem. Pharmacol. 31, 3801-3806[CrossRef][Medline] [Order article via Infotrieve]
  50. Ohtaki, S., Nakagawa, H., Nakamura, M., and Yamazaki, I. (1982) J. Biol. Chem. 257, 761-766[Abstract/Free Full Text]
  51. Chen, Y.-R., Sturgeon, B. E., Gunther, M. R., and Mason, R. P. (1999) J. Biol. Chem. 274, 24611-24616[Abstract/Free Full Text]
  52. Finkelstein, E., Rosen, G. M., and Rauckman, E. J. (1980) Arch. Biochem. Biophys. 200, 1-16[CrossRef][Medline] [Order article via Infotrieve]
  53. Hosoya, T., Sakurada, J., Kurokawa, C., Toyoda, R., and Nakamura, S. (1989) Biochemistry 28, 2639-2644[CrossRef][Medline] [Order article via Infotrieve]
  54. Ortiz de Montellano, P. R. (1987) Acc. Chem. Res. 20, 289-294[CrossRef]
  55. Ortiz de Montellano, P. R., David, S. K., Ator, M. A., and David, T. (1988) Biochemistry 27, 5470-5476[CrossRef][Medline] [Order article via Infotrieve]
  56. Ator, M. A., David, S. K., and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 262, 14954-14960[Abstract/Free Full Text]
  57. Jung, M. J., and Metcalf, B. W. (1975) Biochem. Biophys. Res. Commun. 67, 301-306[CrossRef][Medline] [Order article via Infotrieve]

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