Title: A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels Jaganathan L, Boopathy R Ref: BMC Biochem, 1:3, 2000 : PubMed
BACKGROUND: In vertebrates, two types of cholinesterases exist, acetylcholinesterase and butyrylcholinesterase. The function of acetylcholinesterase is to hydrolyse acetylcholine, thereby terminating the neurotransmission at cholinergic synapse, while the precise physiological function of butyrylcholinesterase has not been identified. The presence of cholinesterases in tissues that are not cholinergically innervated indicate that cholinesterases may have functions unrelated to neurotransmission. Furthermore, cholinesterases display a genuine aryl acylamidase activity apart from their predominant acylcholine hydrolase activity. The physiological significance of this aryl acylamidase activity is also not known. The study on the aryl acylamidase has been, in part hampered by the lack of a specific method to visualise this activity. We have developed a method to visualise the aryl acylamidase activity on cholinesterase in polyacrylamide gels. RESULTS: The o-nitroaniline liberated from o-nitroacetanilide by the action of aryl acylamidase activity on cholinesterases, in the presence of nitrous acid formed a diazonium compound. This compound gave an azo dye complex with N-(1-napthyl)-ethylenediamine, which appeared as purple bands in polyacrylamide gels. Treating the stained gels with trichloroacetic acid followed by Tris-HCl buffer helped in fixation of the stain in the gels. By using specific inhibitors for acetylcholinesterase and butyrylcholinesterase, respectively, differential staining for the aryl acylamidase activities on butyrylcholinesterase and acetylcholinesterase in a sample containing both these enzymes has been demonstrated. A linear relationship between the intensity of colour developed and activity of the enzyme was obtained. CONCLUSIONS: A novel method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels has been developed.
Title: Distinct Effect of Benzalkonium Chloride on the Esterase and Aryl Acylamidase Activities of Butyrylcholinesterase Jaganathan L, Boopathy R Ref: Bioorg Chem, 28:242, 2000 : PubMed
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.
Title: Interaction of Triton X-100 with acyl pocket of butyrylcholinesterase: effect on esterase activity and inhibitor sensitivity of the enzyme Jaganathan L, Boopathy R Ref: Indian J Biochem Biophys, 35:142, 1998 : PubMed
The effect of non-ionic detergents like Triton X-100, Lubrol PX, Brij 35 and Tween 80 on the esterase activity and inhibitor sensitivity of human serum butyrylcholinesterase (BCHE) were studied. The results showed that though BCHE is not a detergent dependent enzyme, the esterase activity and inhibitor sensitivity of it can be modulated by the presence of detergents. All the detergents caused a marginal activation of the esterase activity. The presence of Lubrol PX, Brij 35 or Tween 80 did not affect the 50% molar inhibition concentration (IC50) of the inhibitors tested. But in the presence of Triton X-100 the IC50 values were increased for neostigmine, eserine and tetraisopropylpyrophosphoramide (acylation site interacting inhibitors), whereas for inhibitors like ethopropazine, imipramine and procainamide (choline binding pocket specific inhibitors) the IC50 values were unaltered. In addition, in the presence of Triton X-100 the bimolecular reaction constant for phosphorylation reaction (ki) of BCHE for the acyl pocket specific tetraisopropylpyrophosphoramide was reduced. Triton X-100 partially protected BCHE against this tetraisopropylpyrophosphoramide inactivation. These results indicate that Triton X-100 by interacting with the acyl pocket hydrophobic region is able to activate the esterase activity of BCHE. Further it reduces the capacity of the enzyme to react with inhibitors that inactivate it by interacting with the serine residue of the acylation site.
Title: Hydrophobicity on Esterase Activity of Human Serum Cholinesterase Jaganathan L, Padmalatha K, Revathi G, Boopathy R Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:123, 1995 : PubMed
