Human butyrylcholinesterase components differ in aryl acylamidase activity

Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.     

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.     

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.     

The expression of cholinesterases in human renal tumours varies according to their histological types

The change in the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in neoplastic colon and lung prompted us to study the possible effect of cancer on the expression of cholinesterases (ChEs) in kidney. Samples of papillary renal cell carcinoma (pRCC), conventional RCC (cRCC), chromophobe RCC (chRCC) and renal oncocytoma (RON), beside adjacent non-cancerous tissues, were analyzed. In pRCC both AChE and BuChE activities were statistically increased; in cRCC and chRCC only AChE activity increased and in RON neither AChE nor BuChE activities were affected. Abundant amphiphilic AChE dimers (G(2)(A)) and fewer monomers (G(1)(A)) were identified in healthy kidney as well as in all tumour classes. Incubation with PIPLC revealed glycosylphosphatidylinositol in AChE forms. BuChE is distributed between principal G(4)(H), fewer G(1)(H), and much fewer G(4)(A) and G(1)(A) species. RT-PCR showed similar amounts of AChE-H, AChE-T and BuChE mRNAs in healthy kidney. Their levels increased in pRCC but not in the other tumour types. The data support the idea that, as in lung tumours, in renal carcinomas expression of ChE mRNAs, biosynthesis of molecular components and level of enzyme activity change according to the specific kind of cell from which tumours arise.     

On the active site for hydrolysis of aryl amides and choline esters by human cholinesterases

Cholinesterases, in addition to their well-known esterase action, also show an aryl acylamidase (AAA) activity whereby they catalyze the hydrolysis of amides of certain aromatic amines. The biological function of this catalysis is not known. Furthermore, it is not known whether the esterase catalytic site is involved in the AAA activity of cholinesterases. It has been speculated that the AAA activity, especially that of butyrylcholinesterase (BuChE), may be important in the development of the nervous system and in pathological processes such as formation of neuritic plaques in Alzheimer's disease (AD). The substrate generally used to study the AAA activity of cholinesterases is N-(2-nitrophenyl)acetamide. However, use of this substrate requires high concentrations of enzyme and substrate, and prolonged periods of incubation at elevated temperature. As a consequence, difficulties in performing kinetic analysis of AAA activity associated with cholinesterases have hampered understanding this activity. Because of its potential biological importance, we sought to develop a more efficient and specific substrate for use in studying the AAA activity associated with BuChE, and for exploring the catalytic site for this hydrolysis. Here, we describe the structure-activity relationships for hydrolysis of anilides by cholinesterases. These studies led to a substrate, N-(2-nitrophenyl)trifluoroacetamide, that was hydrolyzed several orders of magnitude faster than N-(2-nitrophenyl)acetamide by cholinesterases. Also, larger N-(2-nitrophenyl)alkylamides were found to be more rapidly hydrolyzed by BuChE than N-(2-nitrophenyl)acetamide and, in addition, were more specific for hydrolysis by BuChE. Thus, N-(2-nitrophenyl)alkylamides with six to eight carbon atoms in the acyl group represent suitable specific substrates to investigate further the function of the AAA activity of BuChE. Based on the substrate structure-activity relationships and kinetic studies, the hydrolysis of anilides and esters of choline appears to utilize the same catalytic site in BuChE.     

Interaction of metal chelators with different molecular forms of acetylcholinesterase and its significance in Alzheimer's disease treatment

The peripheral anionic site (PAS) of acetylcholinesterase (AChE) is involved in amyloid beta (Aβ) peptides aggregation of Alzheimer's disease (AD). AChE exhibits an aryl acylamidase (AAA) activity along with the well known esterase activity. Numerous studies have reported the beneficiary effect of metal chelators in AD treatment. Hence, a comparative study on the effect of metal chelators on both the esterase and AAA activity of AChE globular (G4 and G1) molecular forms was performed. The inhibitory effect of 1,10‐phenanthroline was high towards AChE esterase activity. The corresponding IC₅₀ values for esterase activity of G4 and G1‐form was 190 µM and 770 µM and for AAA activity it was 270 µM and 2.74 mM, respectively. Kinetic studies on both forms of AChE show that 1,10‐phenanthroline inhibits esterase in competitive and AAA activity in non‐competitive manner. Protection studies further revealed that the nature of 1,10‐phenanthroline inhibition on AChE is through its direct binding to protein rather than its metal chelation property. Molecular docking studies shows orientation of 1,10‐phenathroline in the PAS through hydrophobic interactions with the PAS residues (Trp286, Tyr124 and Tyr341) and hydrogen bonding with Phe295. Further molecular dynamics simulation of “hAChE‐1,10‐phenanthroline” complex revealed that both hydrogen and hydrophobic interaction contribute equally for 1,10‐phenanthroline binding to hAChE. Such an interaction of 1,10‐phenanthroline on PAS may hinder “AChE‐Aβ peptide” complex formation. Hence, 1,10‐phenanthroline can act as a lead molecule for developing drug(s) against AD ailment with dual functions namely, anti‐cholinesterase and anti‐amyloid aggregation potency in a single chemical entity. Proteins 2013. Proteins 2013; 81:1179–1191. © 2013 Wiley Periodicals, Inc.     

Diisopropylfluorophosphate-sensitive aryl acylamidase activity of fatty acid free human serum albumin

Butyrylcholinesterase in human plasma and acetylcholinesterase in human red blood cells have aryl acylamidase activity toward o-nitroacetanilide, hydrolyzing the amide bond to produce o-nitroaniline and acetate. People with a genetic variant of butyrylcholinesterase that had no detectable activity with butyrylthiocholine, nevertheless had aryl acylamidase activity in their plasma. To determine the source of this aryl acylamidase activity we tested fatty acid free human albumin for activity. We found that albumin had aryl acylacylamidase activity and that this activity was inhibited by diisopropylfluorophosphate. Since the esterase activity of albumin is also inhibited by diisopropylfluorophosphate, and since it is known that diisopropylfluorophosphate covalently binds to Tyr 411 of human albumin, we conclude that the active site for aryl acylamidase activity of albumin is Tyr 411. Albumin accounts for about 10% of the aryl acylamidase activity in human plasma.     

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.     

The aryl acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain

The appearance of cholinergic trait often precedes synaptogenesis, indicating the involvement of cholinesterase proteins in nervous system development, particularly so acetylcholinesterase (AChE). In addition to AChE's acclaimed esterase activity, its lesser known non-cholinergic functions have gained much attention, because of AChE protein expression in areas other than cholinergic innervations; one such function could be exerted by its associated aryl acylamidase (AAA) activity. In this study, an attempt has been made in profiling esterase and AAA activities of AChE at different developmental stages of the chick embryo, e.g. at embryonic day 6 (E6), E9, E12, E15 and E18. AAA activity showed a correlated expression with esterase activity at all stages, but the relative ratios of AAA to esterase activity were higher at younger stages. The inhibition of AAA activity was shown to be more sensitive towards Huperzine, Donepezil whereas inhibition of esterase activity was sensitive to Tacrine and DFP. Remarkably, the major Alzheimer drugs- Huperzine and Donepezil, much more strongly inhibited AAA activity of AChE at younger developmental stages whose IC50 values are 0.01 μM and 0.1 μM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 μM. Since in Alzheimer's disease (AD), embryonic forms of AChE have been reported to reappear, a possible role of AAA activity in the pathogenesis of AD should be considered.     

Generation of a monoclonal antibody that has reduced binding activity to VX-inactivated butyrylcholinesterase (BuChE) compared to BuChE by phage display

Organophosphate (OP) nerve agents are known as the most toxic chemical warfare agents that act by inhibiting the enzyme acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Because BuChE is present at a level of about 3,900 times higher than AChE in plasma, most OP agents first react with BuChE in plasma, suggesting that OP-inactivated BuChE (OP-iBuChE) may act as a biomarker of OP exposure. In this study, we generated an anti-BuChE monoclonal antibody (mAb) that has reduced binding activity to VX-inactivated BuChE compared to native BuChE by phage display. We performed subtractive biopanning of a synthetic human Fab library against native BuChE and soman-iBuChE or VX-iBuChE. As the results, we isolated four Fab clones that showed differential binding activities to VX-iBuChE and native BuChE in ELISAs. To confirm the antigen-binding specificity of the selected clones, the Fabs were converted to IgG1s, and the IgG antibodies were expressed in HEK293F cells and purified. One of them (A2) showed approximately 30% reduced binding activity to VX-iBuChE compared to BuChE in a dose-dependent manner, whereas the other three antibodies showed almost the same binding activities to VX-iBuChE and BuChE. In addition, the A2 antibody did not show reduced binding activity to sarin-iBuChE or soman-iBuChE compared to native BuChE. The results indicate that A2 antibody shows reduced binding activity only to VX-iBuChE. A2 antibody may be applied to specific diagnosis of VX exposure.     

Potencies and selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer's disease brain

Eight inhibitors of acetylcholinesterase (AChE), tacrine, bis-tacrine, donepezil, rivastigmine, galantamine, heptyl-physostigmine, TAK-147 and metrifonate, were compared with regard to their effects on AChE and butyrylcholinesterase (BuChE) in normal human brain cortex. Additionally, the IC50 values of different molecular forms of AChE (monomeric, G1, and tetrameric, G4) were determined in the cerebral cortex in both normal and Alzheimer's human brains. The most selective AChE inhibitors, in decreasing sequence, were in order: TAK-147, donepezil and galantamine. For BuChE, the most specific was rivastigmine. However, none of these inhibitors was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate and galantamine inhibited both the G1 and G4 AChE forms equally well. Interestingly, the AChE molecular forms in Alzheimer samples were more sensitive to some of the inhibitors as compared with the normal samples. Only one inhibitor, rivastigmine, displayed preferential inhibition for the G1 form of AChE. We conclude that a molecular form-specific inhibitor may have therapeutic applications in inhibiting the G1 form, which is relatively unchanged in Alzheimer's brain.     

Butyrylcholinesterase activity and molecular components in thymus of healthy and merosin-deficient Lama2dy mice

The laminin-alpha2 chain, referred to as merosin, forms part of the laminin-2 heterotrimer (alpha2beta1gamma1), which is principally expressed in the basement membrane of muscle. Nearly half of patients suffering from congenital muscular dystrophy (CMD) have abnormalities in the laminin-alpha2 chain (LAMA2) gene, and the merosin-deficient Lama2dy mouse shows CMD. The expression of merosin in thymus, the abnormalities in the gland of Lama2dy mice, and the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in thymus prompted us to study the possible effects of the deficiency of merosin on thymus BuChE. We found that, while AChE activity decreased by approximately 50% in merosin-deficient thymus, the deficiency had little effect on BuChE activity. About 65% of thymus BuChE activity was extracted with a saline buffer and 30% with 1% Triton X-100. Sedimentation analyses and phenyl-agarose chromatography showed that thymus contained amphiphilic BuChE monomers (G(1)(A),44%) and dimers (G(2)(A),33%), and hydrophilic tetramers (G(4)(H),23%). Binding assays with various plant lectins revealed differences between the oligoglycans linked to BuChE tetramers and lighter components. The deficiency of merosin had no effect on the biosynthesis of thymus BuChE as judged by the lack of major changes between control and Lama2dy mice thymuses in the distribution of BuChE molecules and the level of lectin binding. The detoxifying action of BuChE, its role as a backup to AChE, and the relevance of the cholinergic dialogue between T cells and stromal cells for T lymphocyte proliferation, maturation and survival support a physiological function for BuChE in thymus.     

Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogeneous enzyme.

The asymmetric (20S) form of acetylcholinesterase (AChE) in 1-day-old chick muscle is a hybrid enzyme containing both AChE (110 kd) and butyrylcholinesterase (BuChE, 72 kd) catalytic subunits. However, we now report that the asymmetric AChE extracted or immunopurified from older adult chicken muscles, where it is the endplate form, shows a progressive developmental loss of the BuChE subunit and its activities, centred around 4 weeks of age, while the AChE and collagenous subunits remain. In confirmation, using differential labelling and co-sedimentation it was shown that the hybrid 20S AChE/BuChE form of 1-day chick muscle is gradually and completely replaced during muscle maturation by a 21.3S form, also collagen-tailed but otherwise homogeneous in AChE catalytic subunits. Two other changes occur concomitantly. Firstly, the AChE catalytic subunit of the adult form has a lower apparent mol. wt in gel electrophoresis, by 5 kd, than the same subunit in the 1-day hybrid enzyme; this difference does not reside in the carbohydrate attachments. Secondly, the collagen tail changes, in that some conformation-dependent epitopes on it disappear in the same period. Hence, a major reorganization of the asymmetric AChE, involving all three types of subunit, occurs in the course of muscle development.     

Enantiomer effects of huperzine A on the aryl acylamidase activity of human cholinesterases

1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are serine hydrolase enzymes that catalyze the hydrolysis of acetylcholine.2. (–) Huperzine A is an inhibitor of AChE and is being considered for the treatment of Alzheimer's disease.3. In addition to esterase activity, AChE and BuChE have intrinsic aryl acylamidase activity.4. The function of aryl acylamidase is unknown but has been speculated to be important in Alzheimer pathology.5. Kinetic effects of (–) huperzine A and ( ±)$ huperzine A on the aryl acylamidase activity of human cholinesterases were examined.6. (–) Huperzine A inhibited the aryl acylamidase activities of both AChE and BuChE.7. (±) Huperzine A inhibited this function in AChE but stimulated BuChE aryl acylamidase suggesting that the (+) enantiomer is a powerful activator of this enzyme activity.8. The two huperzine enantiomers may prove to be useful tools to examine the function of aryl acylamidase activity, including its role in Alzheimer pathology.     

THE ACETYLCHOLINESTERASE AND PSEUDO- CHOLINESTERASE CONTENTS OF HUMAN PLACENTA AT TERM

The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities of human term placentas have been studied. Perfusion of placentas indicated that most of the AChE and all the BuChE content of the human placenta are due to its blood content. By comparison, rabbit livers, studied similarly, retained most of their contents of both enzymes following perfusion. Histochemical examination or well-perfused placentas suggests that trapped intact erythrocytes and the stroma of hemolyzed red cells are the probable source of ‘placental’ AChE reported previously.     

A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters

Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.     

Divergent Regulation of Acetylcholinesterase and Butyrylcholinesterase in Tissues of the Rat

Abstract: Investigating the possibility that acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague-Dawley, Fischer-344, and Wistar-Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague-Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100-fold range of BuChE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer-344 or Wistar-Furth rats. Experiments with internal standards of partially purified AChE and BuChE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue-specific. By contrast, BuChE activity showed highly significant correlations among the majority of the tissues examined in the Sprague-Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body-wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar-Furth rats showed significant correlations of BuChE activity. In general, AChE and BuChE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.     

Inhibition of esterase and amidase activities of alpha- and beta-thrombin in the presence of antithrombin III and heparin

Inhibition of the esterase and amidase activities of bovine alpha- and beta-thrombin in the presence of antithrombin III and heparin has been studied. It was found that both the esterase and amidase activities of alpha-thrombin were inhibited by antithrombin III and the reactions were accelerated by heparin. The inhibition of amidase and esterase activities of beta-thrombin by antithrombin III has also been demonstrated. Heparin however did not increase the rate of inactivation of the enzyme.     

Chloramphenicol and Ampicillin-Induced Changes in Rat Hepatic Esterase and Amidase Activities

The influence of ampicillin and chloramphenicol administered intraperitoneallysingly or in combination on the protein content and the activities of hepaticsterase and amidase have been investigated in rats. The results have beencompared to the effects of phenobarbitone (inducer) andp-nitrophenyl-phosphate (inhibitor) of hepatic hydrolases.Ampicillin pretreatment reduced protein level and amidase activity by3.5% each but caused a significant increase (8.1%) in total esteraseactivity compared to controls. Chloramphenicol treatment caused an overalldecrease in protein level, esterase and amidase activities respectively by11%, 11%, and 35% over controls.Combined administration of both drugs resulted in a decrease in protein,esterase and amidase activities by 11.5%, 12.5%, and 41.2% respectively,thus mimicking the effects obtained with chloramphenicol alone.The changes induced by administration of the drugs particularly incombination on the constituent enzymes of rat hepatic hydrolases may affectthe ability of the body to deal with exposure to environmental chemicals ifextrapolated to man.