Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors.

The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.

Overexpression of the endoplasmic reticulum 60 protein ER-60 downregulates apoB100 secretion by inducing its intracellular degradation via a nonproteasomal pathway: evidence for an ER-60-mediated and pCMB-sensitive intracellular degradative pathway.

Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway.

[Identification of catalytically active groups in inulinase from Bacillus polymyxa 722]. / Identifikatsiia kataliticheski aktivnykh grupp inulinazy Bacillus polymyxa 772.

Inulinase from Bacillus polymyxa 722 hydrolyzing a polyfructosan inulin was studied. The dependence of inulinase activity on pH, measurements of pK value, calculation of ionization heat, photoinactivation with methylene blue, and inhibition with p-chloromercuribenzoate suggest that the active center of this enzyme contains imidazole and sulfhydryl groups. A possible mechanism underlying cleavage of beta-2,1-fructoside bonds in the inulin molecule with inulinase is considered.

Studies on trypsin-modified bovine and human lens acylpeptide hydrolase.

Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203--204 of the native acylpeptide hydrolase results in the formation of a 55 kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens beta- and gamma-crystallins as substrates. SDS--PAGE analysis indicated that the beta B2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10--26 kDa). No cleavage of the gamma-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55 kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS--PAGE analysis followed by fluorography of ((3)H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55 kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH(2)--the amino acid sequence 200--207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native protein--was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55 kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate.

The mechanism of contraction by 2-chloroadenosine in cat detrusor muscle cells.

PURPOSE: Four adenosine receptors (ARs), designated A1AR (A1 adenosine receptor), A2aAR (A2a adenosine receptor), A2bAR (A2b adenosine receptor), and A3AR (A3 adenosine receptor), have been cloned from various species, but the contraction mechanism via A1ARs in cat detrusor muscle cell is not well known. MATERIALS AND METHODS: We examined the cellular mechanism using an A1AR agonist 2-chloroadenosine (2-CA) in cat detrusor cell isolated by enzymatic digestion. To examine which phospholipase mediates the contraction, we used phospholipase inhibitors. RESULTS: The adenosine analog potency order is R-N6-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarbosamine adenosine (NECA) > 2-chloroadenosine (2-CA) > S-N6-phenylisopropyladenosine (S-PIA). The ratio of equi-effective concentrations of R-PIA/S-PIA was 58.2. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM) shifted to the right the concentration-response curves of 2-CA. These results indicate A1ARs mediate 2-CA induced contraction in cat detrusor muscle. G proteins (Gi1, Gi2, Gi3, Go, Gs, and Gq) in cat detrusor muscle were detected by immunoblot analysis. Pertussis toxin (PTX) inhibited 2-CA induced contraction. In permeabilized cells, antibodies against Galphai3 antagonized 2-CA induced contraction, suggesting that the contraction is mediated by Gi3 protein. A phosphatidylinositol-specific phospholipase C (PLC) inhibitor, neomycin, reduced 2-CA induced contraction, but a phospholipase D (PLD) inhibitor, p-chloromercuribenzoic acid, and a phospholipase A2 (PLA2) inhibitor, dimethyl-eicosa-dienoic acid (DEDA), had no effect. We found the presence of the main PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1. 2-CA induced contraction in permeabilized cells was inhibited by PLC-beta3 but not by PLC-beta1 or PLC-gamma1 antibody. These results imply that A1ARs are coupled to PLC-beta3 via PTX-sensitive Gi3 protein. Sr2+ medium and thapsigargin, which replaces intracellular Ca2+ and deplete intracellular calcium stores respectively, inhibited 2-CA induced contraction. CONCLUSIONS: These results suggest that A1ARs mediating 2-CA induced contraction exist in cat detrusor muscle and the contraction depends on a PTX-sensitive Gi3 protein, PLC-beta3 and the release of intracellular Ca2+.

Purification and characterization of cysteine protease from Pleurotus ostreatus.

Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its M(r) was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL.

Structure of the interhelical loops and carboxyl terminus of bacteriorhodopsin by X-ray diffraction using site-directed heavy-atom labeling.

The positions of single amino acids in the interhelical loop regions and the C-terminal tail of bacteriorhodopsin (bR) were investigated by X-ray diffraction using site-directed heavy-atom labeling. Since wild-type bR does not contain any cysteines, appropriate cysteine mutants were produced with a unique sulfhydryl group at specific positions. These sites were then labeled with mercury using the sulfhydryl specific reagent p-chloromercuribenzoate (p-CMB). The cysteine mutants D96A/V101C, V130C, A160C, and G231C were derivatized with labeling stoichiometries of 0.93 +/- 5%, 0.85 +/- 5%, 0.79 +/- 7%, and 0.77 +/- 8%, respectively (Hg per bR). No incorporation was observed with wild-type bR under the same conditions. All mutants and heavy-atom derivatives were fully active as judged by the kinetics of the photocycle and of the proton release and uptake. Moreover, the unit cell dimensions of the two-dimensional P3 lattice were unchanged by the mutations and the derivatization. This allowed the position of the mercury atoms, projected onto the plane of the membrane, to be calculated from the intensity differences in the X-ray diffraction pattern between labeled and unlabeled samples using Fourier difference methods. The X-ray diffraction data were collected at room temperature from oriented purple membrane films at 100% relative humidity without the use of dehydrating solvents. These native conditions of temperature, humidity, and solvent are expected to preserve the structure of the surface-exposed loops. Sharp maxima corresponding to a single mercury atom were found in the difference density maps for D96A/V101C and V130C. Residues 101 and 130 are in the short loops connecting helices C/D and D/E, respectively. No localized difference density was found for A160C and G231C. Residue 160 is in the longer loop connecting helices E and F, whereas residue 231 is in the C-terminal tail. Residues 160 and 231 are apparently in a more disordered and mobile part of the structure.

Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes.

It is increasingly evident that cell swelling is associated with the triggering of many biological processes, including progression of the cell cycle, hormonal response, and gene expression. However, the mechanism by which cell swelling is initially sensed and converted into intracellular signals is still ill-defined. We report here an early event in the detection of cell swelling and initiation of the volume regulatory response in Giardia intestinalis, an ancient representative of the eukaryotic kingdom. Giardial cell swelling, irrespective of the extent, was sensed at a cell volume of 1.06 x isosmotic volume (the threshold volume), at which the transition of the volume regulatory transport system from the 'resting' to the 'open' state occurred. Irreversible modification by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) of reduced thiols affected the threshold volume, but in opposing manners: pCMB increased the threshold volume to 1.14 x and NEM decreased to 0.85 x isosmotic volume. The simple modification of the threshold volume by NEM caused a drastic reduction of giardial cell volume under isosmotic conditions, with a process strikingly similar to the opening of mitochondrial permeability transition pore, a causative event in stress-induced programmed cell death. Substantial evidence supports the hypothesis that modulation of the membrane thiol moieties at the threshold volume, causing the 'all-or-nothing' type of swelling detection, represents the event linking cell swelling to the second messenger systems for volume regulation in present eukaryotes. Pathophysiological implications of alteration of the threshold volume are discussed.

A study of prolyl endopeptidase in bovine serum and its relevance to the tissue enzyme.

Prolyl endopeptidase (PE) belongs to a group of enzymes that specifically recognise the imino acid proline. The characterisation of bovine serum PE was undertaken so that its relationship to its tissue counterparts could be considered. Using various chromatographic methods, PE was partially purified from bovine serum. This preparation was deemed to be enzymatically pure, based on its failure to hydrolyse a wide range of fluorimetric substrates. A native molecular mass of 69.7 kDa was estimated for the enzyme. PE was optimally active at pH 8.0-8.5, demonstrated a preference for phosphate buffer and remained stable over a pH range of 5.0-9.0. A narrowly focused optimal assay temperature of 37 degrees C was evident. Functional reagent studies indicated that this enzyme was a serine protease with a cysteine residue located near or at the active site. The enzyme was also sensitive to heavy metal inhibition. Substrate specificity investigations revealed that the bioactive peptides angiotensin II, bradykinin, luliberin and substance P were hydrolysed by the enzyme preparation, but lower specificities were evident towards these peptides in comparison with the enzyme's tissue counterparts. Specific inhibitor studies, using a range of compounds previously untested against a single PE source, indicated that alpha-ketobenzothiazole was the most effective PE inhibitor, with an IC50 value of 41 pM. In conclusion, the results presented in this paper indicate that bovine serum PE shares many of the characteristics associated with its tissue counterparts, with the exception of its specificity towards certain bioactive peptides.

Thermostable glycerol kinase from Thermus flavus: cloning, sequencing, and expression of the enzyme gene.

The thermostable glycerol kinase (EC 2.7.1.30) gene from Thermus flavus was cloned and expressed in Escherichia coli DH5 alpha. An open reading frame of 1488 bp for the glycerol kinase gene (glpK) starting with an ATG methionine codon was found, which encodes a protein of 496 amino acid residues whose calculated molecular weight is 54,835. The amino acid sequence of T. flavus glycerol kinase is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli. Transformants of E. coli DH5 alpha harboring plasmid pGYK12 with a 1505 bp chromosomal DNA fragment containing the T. flavus glycerol kinase gene showed about 23.8-fold higher glycerol kinase activity than T. flavus.

Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals.

We found that human matrix metalloproteases (MMPs) may be processed from their proenzyme forms (proMMP) to their active forms by two new and unique mechanisms: Firstly, by bacterial proteases such as Pseudomonas elastase and Vibrio cholerae protease, which cleave off the N-terminal autoinhibitory domain (so-called cysteine switch) from proMMPs. The second mechanism depends on free radical generation by activated polymorphonuclear leukocytes (PMNs). In this case, peroxynitrite (ONOO-) or nitrogen dioxide radical (.NO2), the reaction products of either superoxide (O2.-) or molecular oxygen (O2) and nitric oxide (.NO), are the key reactants. Both O2.- and .NO are generated by activated macrophages and PMNs as a result of immunologic responses involving various proinflammatory cytokines. .NO2 or ONOO- seems to interact with a single cysteine residue in the propeptide autoinhibitory domain, or so-called cysteine switch of proMMPs, thus transforming proMMPs into their active conformation. Furthermore, reactive oxygen species are known to inactivate the alpha1-protease inhibitor (alpha1-PI), a potent neutrophil elastase inhibitor in plasma. In addition, we found that such radicals activate MMPs which degrade and inactivate alpha1-PI by proteolysis. Thus, the activation of MMPs, accompanied by the inactivation of alpha1-PI, will bring about enhanced proteolytic damage to the matrix tissues of the infected sites by both MMPs and elastase.

Extracellular dextran-induced p-nitrophenyl-alpha-D-glucoside-hydrolyzing enzyme of Bacillus circulans KA-304: a producer of Schizophyllum commune-lytic enzyme.

p-NP-alpha-D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme, increased remarkably when the bacterium was grown on dextran as a carbon source. It was suggested that the increase of the activity was caused by increases of two major species, alpha-D-glucosidase I and alpha-D-glucosidase II. alpha-D-Glucosidase I, which showed a certain reactivity toward dextran, was isolated from the filtrate (MW 70 kDa, 35-fold, 10% recovery). The enzyme was stable around pH 6.5-7.5 and showed its highest activity at pH 6.5. The enzyme preparation inactivated with p-chloromerucuribenzoic acid recovered its activity by incubating with ditiothereitol. Its substrate specificity suggested that the enzyme was an exo-type enzyme with certain affinity toward alpha-1,6-glucosidic linkage.

Carboxy-terminal truncation of long-tailed amyloid beta-peptide is inhibited by serine protease inhibitor and peptide aldehyde.

The 42/43-residue amyloid beta-peptide (Abeta) is widely believed to play a major role in Alzheimer's disease. The present study shows that the rat brain contains a carboxypeptidase that efficiently deletes three amino acids from Abeta1-43. The carboxypeptidase activity in the brain was completely inhibited by 1 mM phenylmethylsulfonyl fluoride, suggesting the protease is a serine carboxypeptidase. The carboxy-terminal truncation of Abeta1-43 was moderately inhibited by carbobenzoxy-Leu-leucinal, carbobenzoxy-Leu-Leu-leucinal, and carbobenzoxy-Leu-Leu-norvalinal, and weakly by antipain. The present data suggest that the serine carboxypeptidase contributes to the generation of short-tailed Abeta peptides and is important in the intracellular clearance of Abeta1-42/43 in brains.

Purification and characterization of a novel thermostable alpha-L-arabinofuranosidase from a color-variant strain of Aureobasidium pullulans.

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced alpha-L-arabinofuranosidase (alpha-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular alpha-L-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75 degrees C, displayed optimal activity at 75 degrees C and pH 4.0 to 4.5, and had a specific activity of 21.48 mumol min-1. mg-1 of protein against p-nitrophenyl-alpha-L-arabinofuranoside (pNP alpha AF). The purified alpha-L-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The K(m) values of the enzyme for the hydrolysis of pNP alpha AF, arabinan, and debranched arabinan at 75 degrees C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The alpha-L-AFase activity was not inhibited at all by L-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).

Transport properties of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in the rat choroid plexus.

Transport properties of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (DDI) were characterized in the isolated rat choroid plexus. AZT and DDI competitively inhibited the active transport of [3H]benzylpenicillin, a prototypic organic anion, with Ki values of 85.4 +/- 13.1 and 155 +/- 22 microM, respectively. Accumulation of [3H]DDI was against an electrochemical potential via a saturable process (K(m) = 29.7 +/- 4.9 microM, Vmax = 13.5 +/- 2.4 pmol min-1/microL tissue) that was inhibited by metabolic inhibitors (carbonylcyanide p-trifluoromethoxyphenylhydrazone, 10 microM, and rotenone, 30 microM) and sulphydryl reagents (p-chloromercuribenzoic acid, 100 microM, and p-chloromercuribenzenesulphonic acid, 100 microM), but did not require an inwardly directed Na+ gradient. Accumulation of [3H]DDI was inhibited by benzylpenicillin and AZT in a dose-dependent manner, with IC50 values of 91.6 +/- 28.9 and 294 +/- 84 microM, respectively. In contrast, no significant accumulation of [3H]AZT was observed. These results suggest that DDI is transported, at least in part, by the transport system for organic anions located on the rat choroid plexus, whereas AZT is recognized, but not transported by this system.

A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.

Arginase of Bacillus brevis Nagano: purification, properties, and implication in gramicidin S biosynthesis.

An arginase [EC 3.5.3.1] was purified to homogeneous state from a gramicidin S-producing Bacillus brevis Nagano. The enzyme has a molecular weight of about 180,000 on gel filtration. The subunit molecular weight is 32,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is hexameric. The optimum pH is found near 10.0. Mn2+ is essential for its activity and Fe2+, Co2+, Ni2+, and Mg2+ cannot replace Mn2+. The enzyme is highly specific for L-arginine with a K(m) value of 12.8 mM for L-arginine, which is similar to that of liver-type arginase in ureotelic animals. B. brevis arginase is apparently induced by the addition of L-arginine to the glutamate medium. The increased formation of L-ornithine, a constituent amino acid of gramicidin S, by arginase may be involved in the accelerated production of gramicidin S by B. brevis in the presence of L-arginine in the growth medium.

Transport of alpha-ketoisocaproate in neuroblastoma NB-2a cells.

Transport of alpha-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free keto-acid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and beta-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyano-2,2'-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na+].