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PubMed1998

Trypanosoma rangeli sialidase: kinetics of release and antigenic characterization.

The epimastigote stage of Trypanosoma rangeli release a sialidase with a high sialic acid hydrolysis capacity. We demonstrate that sialidase secretion is an active process that is reduced at low temperatures and in the presence of sodium azide. The enzyme is continuously released until certain maximally active concentrations are attained in the BHI culture medium when the parasite density reaches 2-3 x 10(6) cells. When introduced into culture medium already containing such enzyme levels, freshly harvested parasites do not secrete additional sialidase. These findings suggest a self-regulating mechanism and a biological role for the secreted T. rangeli sialidase. The secreted enzyme was purified to homogeneity by fractionation with ammonium sulphate and affinity chromatography. Antibodies raised against the purified molecule recognized antigens of similar molecular weights (73 kDa) in western immunoblotting analyses of T. rangeli and T. cruzi whole cell lysates. No antigenic recognition was recorded against T. cruzi active sialidase/trans-sialidase polypeptides or Clostridium perfringens and Vibrio cholerae commercial sialidases. These observations may indicate the expression of different antigenic domains in T. rangeli, T. cruzi and bacterial sialidases.

Animals↗

PubMedDate not supplied

The intestinal absorption of camptothecin, a highly lipophilic drug, across Caco-2 cells is mediated by active transporter(s).

The intestinal transport properties and kinetics of camptothecin (CPT) through Caco-2 cell monolayers were characterized by determining concentration-, temperature-, and ATP-dependence as well as the effect of selected inhibitors. The absorptive permeability (Peff) of CPT was found to be concentration dependent and saturable (K(m) = 31.2 +/- 6.9 microM) indicating the involvement of a high affinity, low capacity transport system. CPT transport was also temperature dependent and inhibited by sodium azide plus 2-deoxyglucose, which deplete cellular ATP, further suggesting that an active, carrier-mediated transport system contributes to CPT absorption. Based on inhibition studies, the involvement of organic anion and cation transporters was implicated but not conclusively demonstrated. Total CPT transport decreased four fold with increasing pH from 5.5 to 8.5 indicating that CPT lactone contributed more significantly to overall CPT transport than CPT carboxylate. The results of these studies suggest that CPT absorption is mediated by multiple mechanisms including significant passive diffusion and active transport components. Since typical substrates for intestinal carriers are hydrophilic and charged, the involvement of putative absorptive carriers in the transport of CPT is a novel finding that may give insight into the erratic oral bioavailability of CPTs observed in the clinic.

Adenosine Triphosphate↗

PubMed2001

Inactivation of creatine kinase during the interaction of indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals.

Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by indomethacin (IM), an effective drug to treat rheumatoid arthritis and gout, with horseradish peroxidase and hydrogen peroxide (HRP-H2O2). IM inactivated CK during its interaction with HRP-H2O2. Under aerobic conditions, inactivation of CK significantly decreased. CK in rat heart homogenate was also inactivated by IM with HRP-H2O2. When IM was incubated with HRP-H2O2, the maximum absorption of IM at 280 nm rapidly decreased and a new peak at 410 nm occurred with isosbestic points at 260 and 312 nm. In contrast, under anaerobic conditions, the spectral change of IM was almost absent, indicating IM was oxidized to the yellow substance by HRP-H2O2. Adding catalase strongly inhibited the production of yellow substance. Sodium azide also blocked the formation of yellow substance and the inactivation of CK. Electron spin resonance signals of IM carbon-centered radical were detected using 2-methyl-2-nitrosopropane during the interaction of IM with HRP-H2O2 under anaerobic conditions. Oxygen was consumed during the interaction of IM with HRP-H2O2. These results suggest that IM carbon-centered radicals may rapidly react with O2 to generate the peroxyl radicals. Sulfhydryl groups and tryptophane residues of CK decreased during the interaction of IM with HRP-H2O2. Other sulfhydryl enzymes, including alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were also readily inactivated during the interaction with HRP-H2O2. Sulfhydryl enzymes seem to be very sensitive to IM activated by HRP-H2O2.

Alcohol Dehydrogenase↗

PubMed2003

Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting.

The herpes simplex virus type 1 (HSV-1) UL51 gene products are virion-associated phosphoproteins with apparent molecular masses of 27, 29, and 30 kDa in HSV-1-infected cells. In this study, we have investigated the intracellular localization and distribution of UL51 protein both in infected cells and in transfected cells expressing only UL51. We found that this protein colocalized closely with Golgi marker proteins such as the Golgi-58K protein and GM130 in transfected cells expressing only UL51. However, in infected cells, the UL51 protein localized to the juxtanuclear region but only partially colocalized with the Golgi maker proteins. Mutant protein analysis revealed that the N-terminal 15 amino acid residues of the UL51 protein sufficed for this Golgi localization property. The UL51 protein redistributed on addition of brefeldin A. This was prevented by pretreatment with 2-deoxyglucose and sodium azide, which results in ATP depletion, but not by pretreatment with NaF and AlCl(3), which activates heterotrimeric G proteins. Moreover, we found that palmitoylation of the UL51 protein through the N-terminal cysteine at position 9 was necessary for its Golgi localization. Protease digestion analysis suggested that the UL51 protein localized on the cytoplasmic face of the membrane in UL51-transfected cells, while in infected cells it localized mainly to the inside of cytoplasmic vesicles and/or the viral envelope. Transmission immunoelectron microscopy revealed an association of UL51 protein-specific labeling with cytoplasmic virions and also with some membranous structure. We infer from these observations that internalization of UL51 protein into the cytoplasmic vesicle and/or virion may occur in association with viral envelopment in HSV-infected cells.

Amino Acid Sequence↗

PubMed1998

Direct inhibition of the pancreatic beta-cell ATP-regulated potassium channel by alpha-ketoisocaproate.

The ATP-regulated potassium (KATP) channel plays an essential role in the control of insulin release from the pancreatic beta-cell. In the present study we have used the patch-clamp technique to study the direct effects of alpha-ketoisocaproate on the KATP channel in isolated patches and intact pancreatic beta-cells. In excised inside-out patches, the activity of the KATP channel was dose-dependently inhibited by alpha-ketoisocaproate, half-maximal concentration being approximately 8 mM. The blocking effect of alpha-ketoisocaproate was fully reversible. Stimulation of channel activity by the addition of ATP/ADP (ratio 1) did not counteract the inhibitory effect of alpha-ketoisocaproate. In the presence of the metabolic inhibitor sodium azide, alpha-ketoisocaproate was still able to inhibit single channel activity in excised patches and to block whole cell KATP currents in intact cells. No effect of alpha-ketoisocaproate could be obtained on either the large or the small conductance Ca2+-regulated K+ channel. Enzymatic treatment of the patches with trypsin prevented the inhibitory effect of alpha-ketoisocaproate. Based on these observations, it is unlikely that the blocking effect of alpha-ketoisocaproate is due to an unspecific effect on K+ channel pores. Leucine, the precursor of alpha-ketoisocaproate, did not affect KATP channel activity in excised patches. Our findings are compatible with the view that alpha-ketoisocaproate not only affects the beta-cell stimulus secretion coupling by generation of ATP but also by direct inhibition of the KATP channel.

Adenosine Triphosphate↗

PubMed2006

The peroxidative coupling of hemigossypol to (+)- and (-)-gossypol in cottonseed extracts.

Peroxidase(s) present in embryo extracts of Gossypium hirsutum cv. Texas Marker 1 catalyzed a bimolecular coupling of [4-(3)H]-hemigossypol to [4,4'-(3)H(2)]-gossypol. The reaction was dependent on the addition of H(2)O(2) and was inhibited 71-94% by 1 and 10mM sodium azide. The phenolic coupling produced 53% (+)-gossypol and 47% (-)-gossypol in close agreement to the 49% (+)-gossypol and 51% (-)-gossypol found in the intact seed. The nearly racemic mixture of (+)-and (-)-gossypol produced in these embryo extracts can be accounted for by non-enzymatic random coupling of the free radicals of hemigossypol produced by the peroxidase. In contrast, peroxidase reaction mixtures containing crude embryo extracts of G. hirsutum var. marie-galante produced 73% (+)-gossypol and 27% (-)-gossypol. These data from the marie-galante extracts and the fact that these intact seed contain 95% (+)-gossypol suggest a regio-stereoselective bimolecular coupling of hemigossypol to gossypol. The development of the peroxidative coupling of hemigossypol to gossypol in maturing seed of G. hirsutum cv. Texas Marker 1 was correlated to the formation of gossypol and suggests that peroxidative coupling of hemigossypol contributes to gossypol biosynthesis.

Chromatography, High Pressure Liquid↗

PubMed2005

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): IX. Evaluation of the mutagenic potential of synthesized L-valyl-L-prolyl-L-proline in the Salmonella-Escherichia coli/microsome, incorporation assay.

The objective of this study was to assess the mutagenic potential of a synthesized tripeptide, L-valyl-L-prolyl-L-proline (VPP), to induce mutational changes in Salmonella typhimurium LT2 strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2uvrA in the classical Ames test protocol. Bacteria were exposed to plate concentrations of VPP of 0, 156.2, 312.5, 625, 1250, 2500, and 5,000 microg/plate in distilled water, in the presence and absence of Aroclor 1254-induced rat liver homogenate preparation (S9). Positive-control agents included sodium azide (TA100 and TA1535); 2-aminoanthracene (TA98, TA100, TA1535, TA1537, and WP2uvrA); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); and N-ethyl-N'-nitro-N-nitrosoguanidine (WP2uvrA) in DMSO. Incubations were conducted at 37 degrees C for about 48 h then revertant colonies were counted. All positive-control agents were consistently and unequivocally positive, but there was no evidence that VPP induced increases in the incidences of revertant colonies in any bacterial strain with and without metabolic activation. These findings were replicated in a second, confirmatory test performed with and without S9. The results of the experiments revealed no treatment-associated changes in the incidence of revertant colonies in any bacterial strain tested. These results support a conclusion that, under the experimental conditions described, there is no evidence that VPP possesses mutagenic potential.

Animals↗

PubMed1999

Tyrosine and calcium/calmodulin kinases are common signaling components in the generation of reactive oxygen species in human lymphocytes.

This study examined the signaling mechanism involved in the generation of reactive oxygen species (ROS) in human lymphocytes activated by formyl-Met-Leu-Phenylalanine (fMLP; 200 nmol/L) or phorbol-myristate-acetate (PMA; 100 nmol/L). ROS were monitored spectrophotometrically using dichlorofluorescin diacetate. fMLP and PMA significantly increased ROS above the control levels (p<0.05 and 0.001, respectively). These increases were significantly inhibited by catalase, sodium azide, and dimethylsulfoxide but not by superoxide dismutase, suggesting that the ROS apparently included hydrogen peroxide, singlet oxygen and hydroxyl ion but not superoxide anion. PMA-induced responses were reduced by tyrphostin (p<0.01), ST-638 (p<0.05), KN-62 (p<0.001), bisindolylmaleimide (p<0.001), RO-31-8220 (p<0.001), and by LY-83583 (p<0.001), suggesting significant involvement of tyrosine kinase, calcium/calmodulin kinase II, protein kinase C and guanylyl cyclase. fMLP-induced responses were significantly reduced by only tyrphostin (p<0.001), ST-638 (p<0.05), and KN-62 (p<0.01). The results show that tyrosine kinase and calcium/calmodulin kinase II are common signalling components in the production of reactive oxygen species in activated lymphocytes.

Aminoquinolines↗

PubMed2006

Motor stimulant effects of ethanol injected into the substantia nigra pars reticulata: importance of catalase-mediated metabolism and the role of acetaldehyde.

A series of experiments was conducted to investigate the locomotor effects of local injections of ethanol and the ethanol metabolite, acetaldehyde, into substantia nigra pars reticulata (SNr). Infusions of ethanol into SNr resulted in a dose-related increase in locomotor activity, with maximal effects at a dose of 1.4 micromol. Ethanol injected into a control site dorsal to substantia nigra failed to stimulate locomotion, and another inactive site was identified in brainstem areas posterior to substantia nigra. The locomotor effects of intranigral ethanol (1.4 micromol) were reduced by coadministration of 10 mg/kg sodium azide, a catalase inhibitor that acts to reduce the metabolism of ethanol into acetaldehyde in the brain. SNr infusions of acetaldehyde, which is the first metabolite of ethanol, also increased locomotion. Taken together, these results indicate that SNr is one of the sites at which ethanol and acetaldehyde may be acting to induce locomotor activity. These results are consistent with the hypothesis that acetaldehyde is a centrally active metabolite of ethanol, and provide further support for the idea that catalase activity is a critical step in the regulation of ethanol-induced motor activity. These studies have implications for understanding the brain mechanisms involved in mediating the ascending limb of the biphasic dose-response curve for the effect of ethanol on locomotor activity.

Acetaldehyde↗

PubMed2002

A correlative study on antimutagenic and chemopreventive activity of Acacia auriculiformis A. Cunn. and Acacia nilotica (L.) Willd. Ex Del.

The present study provides a correlation of the antimutagenic and chemopreventive activity of the barks of two commonly observed plants viz. Acacia auriculiformis and Acacia nilotica. We used the Ames antimutagenicity assay and the mouse mammary gland organ culture (MMOC) model. The plants were extracted with organic solvents to obtain chloroform fractions and acetone extracts. The antimutagenic activity was determined in two different strains using both direct-acting [4-nitro-o-phenylenediamine (NPD) or sodium azide] and indirect-acting [2-aminofluorene (2AF)] mutagens. The anticarcinogenic activity was evaluated based on the development of preneoplastic lesions in response to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The results showed that the activity resulting from the 2AF mutagen was selectively greater than the activity from the direct-acting mutagens. Moreover, in general, acetone extracts were more potent in suppressing mutagenesis than the chloroform extracts. The antimutagenicity results obtained with extracts using the 2AF--TA100 system were comparable to the chemopreventive results with DMBA-induced mammary lesions. The order of activity in both tests was A. nilotica > A. auriculiformis. These results exhibited a good correlation between the antimutagenesis assay and the MMOC model, suggesting that these plants may contain active chemopreventive agents.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

[A new method for the intravital assessment of the functional activity of the membrane transporters performing the efflux of antitumor preparations from the cells of solid tumor specimens].

A new method for intravital assessment of the functional activity of anticancer drug efflux transporters in intact solid tumor specimens was developed. The method is based on the well-known approach to the transporter functional evaluation by intracellular accumulation of antitumor drugs and particularly the anthracycline antibiotic doxorubicin (Dox). The main new point of the method providing investigation of intact solid tumor specimens which markedly simplified the procedure is the fact that the intratissue and intracellular accumulation of Dox is determined not by the level of the drug in the tissue but by its fluorescence decrease in the incubation medium. To assess just the intracellular content of Dox and to estimate the transporter functional activity, investigation of the influence of membrane transporter inhibitors such as verapamil (P-gp inhibitor) and sodium azide (inhibitor of all the energy-dependent ABC transporters) on the drug fluorescence decrease in the incubation medium is stipulated. The validity of such an approach was experimentally proved with the specimens of the Ehrlich solid tumor transplants in mice (a sensitive variant of the tumor and the tumor with induced drug resistance). Biopsy specimens of human breast tumors were investigated with the new method and functional activity of various efflux transporters was revealed: (1) only P-gp, (2) both P-gp and other ABC transporters, (3) only transporters different from P-gp, (4) no functional activity of efflux transporters. The main trends of the further investigation of efflux transporter functional activity in human solid tumors possible for the first time with the use of the new method are defined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Saturable transport of H2-antagonists ranitidine and famotidine across Caco-2 cell monolayers.

The purpose of this study was to investigate the mechanism by which the H2-antagonists ranitidine and famotidine interacted with the paracellular space during their transport across Caco-2 cell monolayers. Transport experiments with ranitidine and famotidine across Caco-2 cell monolayers were performed to determine the apical-to-basolateral flux at various concentrations. Kinetic analysis of the transport data showed that ranitidine and famotidine were transported by both saturable and nonsaturable processes. Na+, K+-ATPase inhibitor ouabain and metabolic inhibitors sodium azide + 2-deoxy-D-glucose did not affect ranitidine transport, suggesting that the active transport was not involved. Famotidine and some other guanidine-containing compounds, e.g., guanethidine, Arg-Gly, L-arginine methyl ester, and L-argininamide, inhibited the transport of ranitidine, whereas other guanidine-containing compounds with an additional negative charge, e.g., L-arginine, did not. 2,4, 6-Triaminopyrimidine (TAP), an inhibitor of paracelluar cationic conductance, also inhibited the transport of both ranitidine and famotidine. On the basis of these results, it is proposed that the saturable transport of ranitidine and famotidine across Caco-2 cell monolayers appears to be via a facilitated diffusion process mediated by the paracellular anionic sites. This mechanism is consistent with the observation that ranitidine and famotidine caused a concentration-dependent increase in transepithelial electrical resistance (TEER) across Caco-2 cell monolayers, presumably by blocking the paracellular anionic sites and thus inhibiting the flux of cations (e.g., Na+).

Biological Transport↗

PubMed2002

Detection of early embryonic development in chicken eggs using visible light transmission.

1. In two separate experiments, the possibility of detecting embryonic development in chicken eggs was assessed using the same spectrophotometric method used to detect blood in Table eggs, using a combination of two wavelengths (577 and 610 nm) of the transmission spectrum. 2. In the first experiment, during the first 10 d of incubation, transmission spectra of 30 Hisex White eggs and 30 Hybro eggs were measured daily. 3. In the second experiment, 292 Hisex White eggs were incubated. Seven groups were randomly assigned. Six received an injection of sodium azide (NaN3) at different times during incubation in order to stop embryonic development, and during the first 12 d of incubation the transmission spectrum was measured daily. The acoustic resonance analysis method was also used on a group of uninjected eggs. 4. In the first experiment, it was possible to detect embryonic development from 120 h of incubation onwards in fertile eggs. In the second experiment changes in light transmission due to embryonic development were detected from 108 h of incubation. Detection of embryonic development using the acoustic resonance analysis method in the second experiment was possible only from 120 h of incubation. 5. It was concluded that the detection of embryonic development using visible light transmission is not directly linked with the formation of blood, but with the formation of sub-embryonic fluid, which takes place from 72 h of incubation onwards. This fluid makes the yolk sac translucent so that absorption of light at 577 nm can be detected.

Absorption↗

PubMed2004

Antimutagenicity of herbal detoxification formula Smoke Shield against environmental mutagens.

Smoke Shield is a formulation designed to reduce smoke related mutagenicity and toxicity in the population. Smoke Shield contains a dual extract of turmeric (Curcuma longa) obtained by supercritical CO2 gas extraction and post-supercritical hydroethanolic extraction together with extracts of green tea and other spices, whose presence synergistically increases the activity of turmeric. In the present study we have shown its antimutagenic activity to various environmental mutagens in vitro and in vivo. Smoke Shield was found to produce significant inhibition of mutagenicity to Salmonella typhimurium induced by sodium azide and 4-nitro-0-phenylenediamine (NPD) at a concentration of 2 mg/plate while inhibition to N-methyl-N-nitro N'nitrosoguanidine was less significant. Inhibition was also found to depend upon the strain which was used. Smoke Shield was found to be more effective against mutagens needing metabolic activation such as 2-Acetamidofluorene (2-AAF) and benzo[a]pyrene. Smoke Shield was also found to significantly inhibit the mutagenicity induced by tobacco extract to Salmonella typhimurium TA102. Smoke Shield was also found to inhibit the urinary mutagenicity of rats treated with the benzo[a]pyrene and tobacco extract. Moreover, Smoke Shield administration was found to inhibit the urinary mutagenicity in smokers. These results indicate that Smoke Shield could inhibit mutagenic response in vitro and in vivo produced by several kinds of mutagens present in our atmosphere.

Animals↗

PubMed2006

Modelling intestinal absorption of salbutamol sulphate in rats.

The objective was to develop a semiphysiological population pharmacokinetic model that describes the complex salbutamol sulphate absorption in rat small intestine. In situ techniques were used to characterize the salbutamol sulphate absorption at different concentrations (range: 0.15-18 mM). Salbutamol sulphate at concentration of 0.29 mM was administered in presence of verapamil (10 and 20 mM), grapefruit juice and sodium azide (NaN3) (0.3, 3 and 6 mM). Different pharmacokinetic models were fitted to the dataset using NONMEM. Parametric and non-parametric bootstrap analyses were employed as internal model evaluation techniques. The validated model suggested instantaneous equilibrium between salbutamol sulphate concentrations in lumen and enterocyte, and the salbutamol sulphate absorption was best described by a simultaneous passive diffusion (ka = 0.636 h(-1)) and active absorption (VMax = 0.726 mM/h, Km = 0.540 mM) processes from intestinal lumen to enterocyte, together with an active capacity-limited P-gp efflux (V'max = 0.678 mM/h, K'm = 0.357 mM) from enterocyte to intestinal lumen. The extent of salbutamol sulphate absorption in rat small intestine can be improved by NaN3, grapefruit juice and verapamil.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Monitoring protein phosphatase 1 isoform levels as a marker for cellular stress.

Reversible protein phosphorylation is a central mechanism regulating many biological functions, and abnormal protein phosphorylation can have a devastating impact on cellular control mechanisms, including a contributing role in neurodegenerative processes. Hence, many promising novel drug development strategies involve targeting protein phosphorylation systems. In this study, we demonstrate that various cellular stresses relevant to neurodegeneration can specifically affect the protein expression levels of protein phosphatase 1 (PP1). PP1 levels were altered upon exposure of PC12 and COS-1 cells to aluminium, Abeta peptides, sodium azide, and even heat shock. Particularly interesting, given PP1's involvement in aging and neurodegeneration, was the consistent decrease in PP1gamma(1) levels in response to stress agents. In fact, alterations in the expression levels of PP1 appear to correspond to an early response of stress induction, that is, before alterations in heat shock proteins can be detected. Our data suggest that monitoring PP1 isoform expression could constitute a useful diagnostic tool for cellular stress, possibly even neurodegeneration. Additionally, our results strengthen the rationale for signal transduction therapeutics and indicate that altering the specific activity of PP1 either directly or by targeting its regulatory proteins may be a useful therapeutic development strategy for the future.

Aluminum Chloride↗

PubMed2001

Effects of cellular ATP depletion on glucose transport and insulin signaling in 3T3-L1 adipocytes.

Glucosamine induced insulin resistance in 3T3-L1 adipocytes, which was associated with a 15% decrease in cellular ATP content. To study the role of ATP depletion in insulin resistance, we employed sodium azide (NaN3) and dinitrophenol (DNP), which affect mitochondrial oxidative phosphorylation, to achieve a similar 15% ATP depletion. Unlike glucosamine, NaN3 and DNP markedly increased basal glucose transport, and the increased basal glucose transport was associated with increased GLUT-1 content in the plasma membrane without changes in total GLUT-1 content. These agents, like glucosamine, did not affect the early insulin signaling that is implicated in insulin stimulation of glucose transport. In cells with a severe 40% ATP depletion, basal glucose transport was similarly elevated, and insulin-stimulated glucose transport was similar in cells with 15% ATP depletion. In these cells, however, early insulin signaling was severely diminished. These data suggest that cellular ATP depletion by glucosamine, NaN3, and DNP exerts differential effects on basal and insulin-stimulated glucose transport and that ATP depletion per se does not induce insulin resistance in 3T3-L1 adipocytes.

3T3 Cells↗

PubMed2000

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance.

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.

Bacterial Proteins↗