Indian Journal of Biochemistry & Biophysics Vol. 40, April 2003, pp. 92-97 Identification of serotonin-sensitive aryl acylamidase activity with cobra venom acetylcholinesterase Ramanna V Rajesh, Aiylam S Balasubramanian and Rathanam Boopathy* Department of Biotechnology, Enzyme Biotech Lab, Bharathiar University, Coimbatore 641 046, India Received 13 May 2002; revised 11 October 2002 Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom anti- body and showed the same heat denaturation profile at 40°C. Further, several potent acetylcholinesterase inhibitors at differ- ent concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in co- bra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observa- tions made with acetylcholinesterase from different sources. The major distribution of acetylcholinesterase (EC. 3.1.1.7; AChE) is across synapses in a variety of nervous systems, where its main role is to terminate impulse transmission by rapid hydrolysis of the neu- rotransmitter acetylcholine. In addition, AChE is also found in erythrocyte membrane, placenta and vascular tissue where its physiological role is not known. Sev- eral in vitro activities have been attributed to AChE, like controlling permeability and transport across non- neuronal membranes. These observations have given rise to the concept of non-cholinergic functions of cholinesterases. Hence a better understanding of cho- linesterases is necessary to clarify additional functions of cholinesterases in development, physiology and disease 1,2 . AChE, in addition to its esterase activity also ex- hibits a serotonin sensitive aryl acylamidase (aryl- acylamide amido hydrolase E. C. 3.5.1.13; AAA) ac- tivity 3-5 . The AAA activity whose physiological func- tion is not known, hydrolyses the artificial substrate o- nitroacetanilide. The serotonin sensitivity of AAA and its identity with AChE have been of interest to many investigators and resulted in speculations about its possible physiological functions 1,2,6 . In the present study, we demonstrate the associa- tion of AAA activity with cobra venom AchE, whose esterase activity has already been reported 7 . The char- acteristics, inhibitor studies, serotonin sensitivity and identity with AChE of the AAA are described. To our knowledge, this is the first report of the AAA activity of cobra venom AChE. Materials and Methods Materials Serotonin (5-hydroxy tryptamine), bis (4-allyl di- methyl ammoniumphenyl) petane-3-one dibromide (BW284c51), tetraisopropylpyrophosphoramide (iso- OMPA), physostigmine (eserine), procainamide, neo- stygmine, Sephadex G-75, acetylthiocholine iodide, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and ven- oms from Cobra (Naja naja), Krait, Saw scaled viper and Russels viper were purchased from Sigma Chemical Company, USA. N-(1-Napthyl)ethyl- enediamine dihydrochloride was procured from E. Merck, Darmstadt, Germany. Anti-snake venom anti- body was purchased from Serum institute of India, Pune. Diisopropylfluorophosphate (DFP) was a kind gift from DRDE, Gwalior. All other chemicals were of analytical grade procured locally. The substrate o-nitroacetanilide was prepared from o-nitroaniline 8 . The final product was recrystallized thrice from hot water and had a melting point of 93-94°C. —————— *Address for correspondence Tel: 091-422-425 716 Fax: 091-422-422 387 E-mail: boopathybiotech@yahoo.com Abbreviations used: AChE, acetylcholinesterase; AAA, aryla- cylamidase activity; DFP, diisopropylfluorophosphate; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); IDA, iminodiacetic acid; BW284c51, bis(4-allyl dimethyl ammoniumphenyl)pentane-3- one-dibromide; iso-OMPA, tetraisopropylpyrophosphoramide.