Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

Toxicological and behavioral study of two potential antibacterial agents:4-chloromercuribenzoic acid and quercetin on Swiss-albino mice.

Global dissemination of carbapenem resistant-Gram negative bacteria (CR-GNB) is supposed to be clinically alarming because it extremely delimits the treatment options against serious infections. 4-Chloromercuribenzoic acid (pCMB) is an efficient metallo-enzyme inhibitor, and quercetin is known for antioxidant, antiviral, anticancer, antimicrobial, and anti-inflammatory activities. These two compounds could be considered as potential candidates for the treatment of CR-GNB mediated infections. Hence, in this study, antibacterial activity of pCMB and quercetin was evaluated against CR-GNB through minimum inhibitory concentration (MIC) determination. Toxicity of pCMB and quercetin was evaluated by LC50 calculation through brine shrimp test (BST) and by investigating hematological, serum biochemical, and histopathological parameters in Swiss-albino mice. Moreover, aggressive-depressive-cognitive behavioral effects of pCMB and quercetin on murine model were evaluated. All the carbapenem resistant isolates (CR-GNB) exhibited MIC values in the range of 4-256 µg/ml, 16-256 µg/ml, and 64-1024 µg/ml for pCMB, quercetin, and meropenem, respectively. BST determined LC50 of pCMB and quercetin at 91.57 ± 0.35 mg/L and 448.45 ± 0.46 mg/L, respectively. Oral administration of low dose of pCMB and quercetin did not induce any significant changes in morphological, behavioral, hematological, serum biochemical, and histopathological parameters among Swiss-albino mice. But, a high dose of pCMB and quercetin exhibited slight toxicity. However, no death was reported for any dosage of pCMB and quercetin. Therefore, pCMB and quercetin might be considered for further investigations on alternative therapeutics to combat against CR-GNB.

[THE EFFECT OF METAL IONES AND SPECIFIC CHEMICAL REAGENTS ON THE ACTIVITY OF ASPERGILLUS FLAVUS VAR. ORYZAE AND BACILLUS SUBTILIS α-AMYLASES].

The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.

New strategy for reversible modulation of protein activity through site-specific conjugation of small molecule and polymer.

A new strategy for accurate and reversible modulation of protein activity via simple conjugation of the sulfhydryl modifier and polymer with the introduced Cys residue in protein was developed in this study. With Escherichia coli inorganic pyrophosphatase (PPase) as a model protein, we used site-directed mutagenesis to generate a mutant PPase (PPC) with a substituted Cys residue at the specific Lys-148 site, which is within a conserved sequence near the active site and exposed to the surface of the PPC for chemical reaction. The site-specific conjugation of the mutated Cys residue in PPC with sulfhydryl modifier p-chloromercuribenzoate (PCMB) and pyridyl disulfide-functionalized poly(2-hydroxyethyl methacrylate) (pHEMA) resulted in obvious decrease or complete loss of the catalytic activity of PPC, due to the conformational change of PPC. Compared with the effect of small molecule modification (PCMB), the pHEMA conjugation led to greater inhibitory effect on protein activity due to the significant change of the tertiary structure of PPC after conjugation. Moreover, the protein activity can be restored to different extents by the treatment with different amount of reductive reagents, which can result in the dissociation between PPC and PCMB or pHEMA to recover the protein conformation. This study provides a new strategy for efficient control of protein activity at different levels by site-specific conjugation of a small molecule and polymer.

An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening.

Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z' factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z' factor of ≥0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC-MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.

Reinvestigation of drugs and chemicals as aquaporin-1 inhibitors using pressure-induced hemolysis in human erythrocytes.

Recently, we have found that pressure-induced hemolysis is enhanced by inhibiting water transport via aquaporin-1 (AQP1), as seen in p-chloromercuribenzoate (pCMB)-treated erythrocytes. So, using this method we reinvestigated the functions as AQP1 inhibitors of drugs and chemicals such as acetazolamide, sodium nitroprusside, tetraethylammonium ions (TEA(+)), and dimethylsulfoxide (DMSO). The values of hemolysis at 200 MPa were almost unaffected by acetazolamide or sodium nitroprusside, decreased by TEA(+), and increased significantly by DMSO. Furthermore, the erythrocytes were exposed to pCMB in the presence of TEA(+) or DMSO. The enhancement effect of pCMB on pressure-induced hemolysis was unaffected by TEA(+) but attenuated by DMSO. Taken together, these results suggest that, of drugs and chemicals examined here, DMSO only is an AQP1 inhibitor, but the effect of DMSO is small compared with pCMB.

[Investigation of functional groups of Cryptococcus albidus alpha-L-rhamnosidase].

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino)propyl]-3-ethylcarbodimide methiodide, EDTA, o-phenantroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the alpha-L-rhamnosidase activity of Cryptococcus albidus has been investigated. The essential role of Ag+ which inhibits the alpha-L-rhamnosidase activity by 72.5% was shown. Rhamnose at 1-5 mM protect the enzyme from the negative effect of Ag(+). It was expected that carboxyl group of C-terminal aminoacid and imidazole group of histidine would participate in the catalytic action of alpha-L-rhamnosidase on the basis of inhibition and kinetic analysis.

Purification and characterization of a novel (R)-imine reductase from Streptomyces sp. GF3587.

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.

Association of the novel non-AT1, non-AT2 angiotensin binding site with neuronal cell death.

We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.

Esterase patterns in species of the triatome bug Rhodnius.

The aim of the present study was to analyse esterase patterns in three triatomine species of Rhodnius genus. Four loci, Est 1, Est 2, Est 3 and Est 4, were found. The corresponding enzymes were characterized as carboxylesterases (E.C. 3.1.1.1) or cholinesterases (E.C. 3.1.1.8) based on inhibitory experiments, using eserine sulphate, malathion, mercury chloride, p-chloromercuribenzoate (pCMB) and iodoacetamide. Low genetic variability was observed: Est 1, Est 2 and Est 3 were monomorphic in Rhodnius domesticus, Rhodnius robustus and Rhodnius neivai, whereas locus Est 4 was polymorphic in the first two species. The UPGMA analysis based on esterase genotypic frequencies indicated greater similarity between R. domesticus and R. robustus when compared with R. neivai. The present study expands our knowledge about genetic variability among triatomines and accords with the hypothesis that R. domesticus is a species derived from R. robustus.

4-Chloromercuribenzoic acid enhances carbapenem sensitivity among pathogenic Gram negative bacteria by altering blaVIM, adeB and ompC expression.

BACKGROUND: Rapid global dissemination of carbapenem resistant Gram negative bacteria (CRGNB) is supposed to be clinically most alarming. Since, p-chloromercuribenzoic acid (pCMB) is a well known metallo-beta-lactamase inhibitor; evaluation of its bactericidal and carbapenem resistance reversing potential would be important. METHODS: In this study, bactericidal and meropenem resistance reversing potential of pCMB was investigated against CRGNB by MIC determination, checkerboard assay, time-kill assay and cellular viability assay. Effect of pCMB on cellular morphology was visualized by Scanning Electron Microscopy. Further, quantitative Real Time-PCR was performed to evaluate effects of pCMB on clinically relevant metallo-beta-lactamases, major efflux pumps and outer membrane proteins expression. RESULTS: pCMB exhibited at least four fold reduced MIC value (2-256µg/ml) than that of meropenem against CRGNB. Moreover, pCMB exhibited synergism with meropenem against 86.06% of CRGNB. MIC of pCMB (16-32µg/ml) could kill upto 99.96% bacteria within 6-8h of dosing. pCMB exerted bactericidal activity by severely disrupting cell wall integrity. Reversal of carbapenemase property of CRGNB by pCMB might have developed through alteration of blaVIM, acrB, mexB and ompk36 expression. CONCLUSIONS: Hence, the current study identified pCMB as a potential bactericidal agent which enhanced meropenem sensitivity by altering blaVIM, acrB, mexB and ompk36 expression.

Cultured ruminal epithelial cells express a large-conductance channel permeable to chloride, bicarbonate, and acetate.

The absorption of short-chain fatty acids (SCFA) from the rumen requires efficient mechanisms for both apical uptake and basolateral extrusion. Previous studies suggest that the rumen expresses a basolateral chloride conductance that might be permeable to SCFA. In order to characterize this conductance in more detail, isolated cultured ruminal epithelial cells were studied with the patch-clamp technique, revealing a whole-cell conductance with p(Cl(-)) approximately p(NO(3) (-)) > p(HCO(3) (-)) > p(acetate(-)) > p(gluconate(-)). Currents could be blocked by diisothiocyanato-stilbene-2,2'-disulfonic acid (1 mmol l(-1) > 100 micromol l(-1)), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (50 micromol l(-1)), niflumic acid (100 micromol l(-1)), and p-chloromercuribenzoate (1 mmol l(-1)). Single-channel conductance was 350 +/- 7 pS for chloride and 142 +/- 7 pS for acetate. Open probability could be fitted with a three-state gating model. We propose a role for this channel in mediating the permeation of chloride, bicarbonate, and acetate across the basolateral membrane of the ruminal epithelium.

High-throughput assays probing protein-RNA interactions of eukaryotic translation initiation factors.

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.

Dietary pectin up-regulates monocaboxylate transporter 1 in the rat gastrointestinal tract.

This work was undertaken to study the effect of pectin feeding on the expression level, cellular localization and functional activity of monocarboxylate transporter 1 (MCT1) in the gastrointestinal tract of rats. The results indicated that MCT1 protein level was significantly increased along the entire length of the gastrointestinal tract of pectin-fed rats in comparison with control animals. Immunohistochemical analysis revealed an increase in MCT1 in the stratified squamous epithelia of the forestomach as well as in the basolateral membranes of the cells lining the gastric pit of the glandular stomach of pectin-fed rats when compared with control animals. The parietal cells, which showed barely any or no detectable MCT1 in the control group, exhibited a strong intensity of MCT1 on the basolateral membranes in pectin-fed rats. In the small intestine of pectin-fed rats, strong immunopositivity for MCT1 was detected in the brush border and basolateral membranes of the absorptive enterocytes lining the entire villi, while in control rats, weak reactivity was detected on the brush border membrane in a few absorptive enterocytes in the villus tip. In the large intestine of control animals, MCT1 was detected on the basolateral membranes of the epithelia lining the caecum and colon. This staining intensity was markedly increased in pectin-fed rats, along with the appearance of strong reactivity for MCT1 on the apical membranes of the surface and crypt epithelia of caecum and colon. Our results also showed that MCT1 co-localizes with its chaperone, basigin (CD147), in the rat gastrointestinal tract, and that the pectin feeding increased the expression of CD147. In vivo functional studies revealed an enhanced acetate absorption in the colon of pectin-fed in comparison with control animals. We conclude that MCT1 is up-regulated along the gastrointestinal tract of pectin-fed rats, which might represent an adaptive response to the increased availability of its substrates.

Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries.

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused 'click' chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine K(i) and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (K(i)=K(i)(')=1.5+/-0.2microM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, K(i)=0.41+/-0.03microM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, K(i)=1.4+/-0.10microM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XV. Agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi).

The water diffusional permeability (P(d)) of red blood cells (RBC) from agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi) was monitored using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. The P(d) (cm s(-1)) values of agile wallaby RBCs were 7.5 x 10(-3) at 25 degrees C, 9 x 10(-3) at 30 degrees C, 11 x 10(-3) at 37 degrees C, and 13 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing sulfhydryl (SH)-modifying reagent p-chloromercuribenzoate (PCMB) on agile wallaby RBCs was investigated. The maximal inhibition was reached in 90 min at 37 degrees C with 2 mmol L(-1) PCMB. The value of maximal inhibition was approximately 63% when measured at 25 degrees C, approximately 52% at 37 degrees C and approximately 45% at 42 degrees C. The lowest value of P(d) (corresponding to the basal permeability to water) was approximately 3 x 10(-3) cm s(-1) at 25 degrees C. For the RBCs from red-necked wallaby (M. rufogriseus) the values of P(d) (cm s(-1)) were 7 x 10(-3) at 25 degrees C, 8 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C, and 12 x 10(-3) at 42 degrees C. Higher values of P(d) (cm s(-1)) were found for the RBCs from Goodfellow's tree kangaroo (D. goodfellowi): 8.5 x 10(-3) at 25 degrees C, 10 x 10(-3) at 30 degrees C, 13 x 10(-3) at 37 degrees C, and 15 x 10(-3) at 42 degrees C. The mean values of the activation energy of water diffusion (E(a,d)) were approximately 25 kJ mol(-1) for RBCs from the agile wallaby and tree kangaroo, respectively, and approximately 23 kJ mol(-1) for RBCs from red-necked wallaby. The values of E(a,d) increased after exposure of agile wallaby RBCs to PCMB, reaching a value of approximately 43-46 kJ mol(-1) when the maximal inhibition of P(d) was achieved.

Characterization of the brain-specific non-AT(1), non-AT(2) angiotensin binding site in the mouse.

In the present study the existence of a non-AT(1), non-AT(2) angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT(1), non-AT(2) angiotensin binding site was similar to the rat brain: Sar(1)-Ile(8)-Ang II > Ang III >or= Ang II > Ang I> p-aminophenylalanine(6) Ang II> CGP42112 >> Ang IV > Ang 1-7 congruent with shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K(i) values >10 microM, while substance P and VIP had K(i) values of approximately 2 microM. The non-AT(1), non-AT(2) angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [(125)I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT(1a), AT(1b), AT(2), Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT(1), AT(2), AT(4) and Ang 1-7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study.

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides.

AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.

Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44.

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.

Identification of a novel non-AT1, non-AT2 angiotensin binding site in the rat brain.

Efforts to protect radiolabeled angiotensins from metabolism during receptor binding assays date back more than 30 years. However, this continues to be a problem. This study focused on the effects of a protease inhibitor, p-chloromercuribenzoate (PCMB), on the binding of (125)I-Ang II to rat brain membranes. Addition of PCMB to the incubation medium revealed a high affinity binding site for (125)I-Ang II in brain membranes (K(d)=1-4 nM) with a greater amount of binding than revealed in previous studies of brain Ang II receptors. Further characterization of this binding, revealed it to be insensitive to inhibition by losartan (an AT(1) receptor antagonist) and PD123319 (an AT(2) receptor antagonist). This non-AT1, non-AT2 binding site was not present in liver or adrenal membranes. It was activated by a limited range of concentrations of PCMB, with maximal activation at 0.3-1 mM. This binding site was equally abundant in cerebral cortex (a brain region with few Ang II receptors) and the hypothalamus (a brain region with abundant Ang II receptors). The binding site was also present in mouse brain, but not mouse liver. The binding site shows high affinity for Ang I, Ang II and Ang III (K(i) approximately 40-100 nM), but lesser affinity for smaller angiotensin fragments and other neuropeptides. This binding site shares some characteristics with the liver cytosolic Ang II binding proteins, later identified as endopeptidases EC 3.4.24.15 and/or EC 3.4.24.16. However, some unique characteristics of this non-AT1, non-AT2 binding site suggest that it may be a novel angiotensin binding substance.