Distinct Effect of Benzalkonium Chloride on the Esterase and Aryl Acylamidase Activities of Butyrylcholinesterase

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from vertebrates, other than their predominant acylcholine hydrolase (esterase) activity, display a genuine aryl acylamidase activity (AAA) capable of hydrolyzing the synthetic substrate o-nitroacetanilide to o-nitroaniline. This AAA activity is strongly inhibited by classical cholinesterase (ChE) inhibitors. In the present study, benzalkonium chloride (BAC), a cationic detergent widely used as a preservative in pharmaceutical preparations, has been shown to distinctly modulate the esterase and AAA activities of BChEs. The detergent BAC was able to inhibit the esterase activity of human serum and horse serum BChEs and AChEs from electric eel and human erythrocyte. The remarkable property of BAC was its ability to profoundly activate the AAA activity of human serum and horse serum BChEs but not the AAA activity of AChEs. Thus BAC seem to preferentially activate the AAA activity of BChEs alone. Results of the study using the ChE active site-specific inhibitor diisopropyl phosphorofluoridate indicated that BAC binds to the active site of ChEs. Furthermore, studies using a structural homolog of BAC indicated that the alkyl group of BAC is essential not only for its interaction with ChEs but also for its distinct effect on the esterase and AAA activities of BChEs. This is the first report of a compound that inhibits the esterase activity, while simultaneously activating the AAA activity, of BChEs. Copyright 2000 Academic Press.     

Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

Characterisation of steryl esterase activities in commercial lipase preparations

Triglycerides, steryl esters, resin acids, free fatty acids and sterols are lipophilic extractives of wood (commonly referred to as pitch or wood resin) and have a negative impact on paper machine runnability and quality of paper. Thus, enzymes capable of modifying these compounds would be potential tools for reducing pitch problems during paper manufacture. In this work, 19 commercial lipase preparations were tested for their ability to degrade steryl esters, which may play a significant role in the formation and stabilisation of pitch particles. Six lipase preparations were shown to be able to degrade steryl esters. Lipase preparations of Pseudomonas sp., Chromobacterium viscosum and Candida rugosa were shown to have the highest steryl esterase activities. The enzymes were able to hydrolyse steryl esters totally in the presence of a surfactant (Thesit). Up to 80% of the steryl esters were degraded in aqueous dispersion. Preliminary characterisation of the enzymatic activities revealed that the lipase preparation of Pseudomonas sp. could be the most potential enzyme in industrial applications. The steryl esterase activity of this preparation was stable over a broad pH range and the enzyme was able to act efficiently at pH 6-10 and at temperatures up to 70 degrees C.     

Kinetic properties of soluble and membrane-bound acetylcholinesterase from electric eel

Using electric eel acetylcholinesterase (AChE) which was either membrane-bound (AChEm) or solubilized (AChEs), similar kinetics were seen in the absence of inhibitor or in the presence of edrophonium, trimethylammonium ion or paraoxon. Thus, both forms of the enzyme appear to behave similarly toward various inhibitors. However, in the presence of a probe sensitive to allosteric effects or changes in membrane fluidity, the two forms exhibit altered behavior. In the presence of F-, the relative rate of substrate hydrolysis by AChEm was reduced more rapidly than with AChEs, whether or not paraoxon was present. When inhibition by paraoxon (10(-7)-10(-4) M) was studied in the presence of F-, AChEs had a Hill coefficient of 1.0, whereas with AChEm the Hill coefficient changed from 0.8 to 1.5.     

Characterization of trimeric acetylcholinesterase from a legume plant, <Emphasis Type="Italic">Macroptilium atropurpureum</Emphasis> Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41–42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs. Kosuke Yamamoto and Yoshie S. Momonoki contributed equally to this work.     

Butyrylcholinesterase-Mediated enhancement of the enzymatic activity of trypsin

1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline.2. Both AChE and BuChE have been shown to copurify with peptidases.3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with -amyloid protein in Alzheimer brain tissues.4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis.5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE.6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.     

人脑及红细胞膜乙酰胆碱酯酶的芳基酰胺酶活性

采用联合亲和层析法从人小脑及红细胞膜中纯化了ＡＣｈＥ,纯化的人脑及红细胞ＡＣｈＥ在ＳＤＳ－ＰＡＧＥ上呈一主带,分子量约为６６０００。人脑ＡＣｈＥ制备酯酶与酰胺酶比活性分别为１２９９与１４３Ｕ／ｍｇ,人红细胞ＡＣｈＥ制备分别为４５８４与７４７Ｕ／ｍｇ。人脑及红细胞ＡＣｈＥ制备的酯酶与酰胺酶活性最适ｐＨ较接近,在ｐＨ７．５－８．０之间,酯酶活性底物ＡＴＣｈ对其芳基酰胺酶活性有抑制作用。ＩＣ_（５０）分别为１０．２×１０~（－３）及３×１０~（－３）ｍｏｌ／Ｌ。梭曼对其酯酶及酰胺酶活性均有明显抑制作用,说明二者均需活性中心丝氨酸参与。     

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.     

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

Identification of Peptidases in Highly Pathogenic vs. Weakly Pathogenic Naegleria fowleri Amebae

Naegleria fowleri, a free‐living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse‐passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba‐CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.     

Acetylcholinesterases from Musca domestica and Drosophila melanogaster Brain Are Linked to Membranes by a Glycophospholipid Anchor Sensitive to an Endogenous Phospholipase

The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol-specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane-bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The lipases uncover an immunological determinant that is found on other glycosylphosphatidylinositol-anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane-bound enzyme migrates faster during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.     

Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.     

Separation of protease activity from acetylcholinesterase of the electric EEL

We examined the protease activity reported to be associated with acetylcholinesterase (AChE) by extensive purification of the electric eel enzyme. Upon edrophonium-Sepharose chromatography of a commercial preparation, a majority of the protease activity was recovered in the effluent with no AChE activity, while a marginal activity was detected in the AChE fraction eluted with edrophonium chloride. Further chromatography of the edrophonium eluate on hydroxyapatite gave partially overlapping peaks of protease and AChE activities. Finally, the protease activity was mostly removed from the AChE fraction by passing through an ovoinhibitor-agarose column. The protease activity in the edrophonium eluate was inhibited by various serine protease inhibitors, but not by AChE inhibitors. These results suggest that the AChE and protease activities are physically separable, and thus that the protease activity, so far reported as intrinsic to AChE, is probably due to contaminants.     

Experimental evidence for interactions between bacterial peptidase and alkaline phosphatase activity in the Baltic Sea

From the observed pattern of aminopeptidase and alkaline phosphatase activities in the Baltic Sea, the question arose whether there is an interaction between the activities of both enzymes. In experiments with 0.8 m filtered seawater, the effects of commercial alkaline phosphatase on bacterial aminopeptidase, the effects of commercial peptidase on bacterial alkaline phosphatase activity (APA), and the effects of proteins, carbohydrates and inorganic nutrients on the activities of both enzymes were investigated.Addition of commercial alkaline phosphatase stimulated bacterial aminopeptidase activity and, similarly, the addition of commercial peptidase increased the APA in bacteria. The proteins, albumin and casein, stimulated aminopeptidase activity and APA simultaneously. Experiments using ammonium and glucose suggested that stimulation of APA by peptidase could be mediated by nitrogen and carbon availability. There were also some indications that stimulation of aminopeptidase activity by alkaline phosphatase functioned by catalysing phosphate release from organic phosphorus compounds.     

Genetic and Environmental Effects on the Expression of Peptidases and Larval Viability in Drosophila Melanogaster

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.     

Location of Peptidases Outside and Inside the Membrane of Streptococcus cremoris

Peptidase activity determinations involving native cells of Streptococcus cremoris and completely disrupted cell preparations, as well as experiments concerned with peptidase activity distribution among cell fractions obtained by a damage-restrictive removal of the cell wall and release of intracellular material, suggest the presence of peptidases with distinguishable locations. Alanyl, leucyl, and prolyl aminopeptidase activities are most likely located in the cell wall-membrane interface, showing no detectable association with the membrane. Lysyl aminopeptidase is present not only in this location, but also as an intracellular enzyme. Endopeptidase activity and glutamate aminopeptidase activity appear to be weakly associated with the membrane. The locations of these two peptidase activities, unlike those of the former aminopeptidase activities, impose a restriction on their expression. Results of experiments concerned with permeabilization of the membrane and findings regarding an effect of the local environment of the enzymes on their pH activity profiles are evaluated and considered as being indicative of the proposed location. The possible implications of these findings with respect to protein utilization during growth of the organism in milk are discussed.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Comparison of the peptidase activity in the oncosphere excretory/secretory products of Taenia solium and Taenia saginata

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.     

Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in the synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase II (DPP II), prolyl endopeptidase (PEP), and collagenase-like peptidase (CLP) activities were higher in knee joint synovial membrane from patients with RA than in that from patients with OA. DPP II and PEP activities in knee joint synovial membrane of patients with RA increased in parallel with the increase in joint fluid volume, whereas DPP IV activity decreased in parallel with the increase in joint fluid volume. These results suggest that these peptidases in the synovial membrane may play some role in immunological disturbances in the joints of patients with RA. Measurement of these peptidases in synovial membrane may be useful in the diagnosis of the severity of local joint inflammation.