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Rathanam Boopathy - One of the best experts on this subject based on the ideXlab platform.
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The significance of Aryl Acylamidase activity of acetylcholinesterase in osteoblast differentiation and mineralization
Molecular and Cellular Biochemistry, 2018Co-Authors: Raj Kumar Chinnadurai, Ponne Saravanaraman, Rathanam BoopathyAbstract:Osteoblast differentiation is an essential event in the developmental process, which is favoured by the production of extra cellular matrix proteins and various enzymes including discrete ones like acetylcholinesterase (AChE). Despite the fact that AChE facilitates osteoblast differentiation, the significance of its catalytic functions [esterase and Aryl Acylamidase (AAA) activities] in the process is yet to be ascertained. In this context, SaOS-2 cell line was used in the present study to implicate the catalytic activities of AChE in process of osteoblast differentiation and mineralization. During differentiation, it was found that the activity of both esterase and AAA increased 1.13 and 1.46 folds respectively, signifying the involvement of catalytic activities of AChE in the process. Inhibition of both the catalytic activities of AChE with edrophonium significantly reduced the amount of mineralization by decreasing the alkaline phosphatase (ALP) activity and expression of differentiation-related genes such as RUNX-2, COL1A, ALP, OC, and OP significantly ( p
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The significance of Aryl Acylamidase activity of acetylcholinesterase in osteoblast differentiation and mineralization.
Molecular and cellular biochemistry, 2017Co-Authors: Raj Kumar Chinnadurai, Ponne Saravanaraman, Rathanam BoopathyAbstract:Osteoblast differentiation is an essential event in the developmental process, which is favoured by the production of extra cellular matrix proteins and various enzymes including discrete ones like acetylcholinesterase (AChE). Despite the fact that AChE facilitates osteoblast differentiation, the significance of its catalytic functions [esterase and Aryl Acylamidase (AAA) activities] in the process is yet to be ascertained. In this context, SaOS-2 cell line was used in the present study to implicate the catalytic activities of AChE in process of osteoblast differentiation and mineralization. During differentiation, it was found that the activity of both esterase and AAA increased 1.13 and 1.46 folds respectively, signifying the involvement of catalytic activities of AChE in the process. Inhibition of both the catalytic activities of AChE with edrophonium significantly reduced the amount of mineralization by decreasing the alkaline phosphatase (ALP) activity and expression of differentiation-related genes such as RUNX-2, COL1A, ALP, OC, and OP significantly (p < 0.05). Inhibition of esterase activity without altering the AAA activity using gallamine significantly increased the level ALP activity and expression of differentiation-associated genes (p < 0.05), thus favouring mineralization. Therefore, this study concludes and confirms that the AAA activity of AChE is actively involved in the process of osteoblast differentiation and mineralization.
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Understanding the molecular mechanism of Aryl Acylamidase activity of acetylcholinesterase - An in silico study.
Archives of biochemistry and biophysics, 2015Co-Authors: Raj Kumar Chinnadurai, Ponne Saravanaraman, Rathanam BoopathyAbstract:Abstract Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and Aryl Acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates ( o -nitroacetanilide, and o -nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900 kJ/mol/nm). Among the two substrates, o -nitrotrifluoroacetanilide required lesser force (380 kJ/mol/nm) than o -nitroacetanilide the (550 kJ/mol/nm) for its binding, thus validating o -nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity.
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High Aryl Acylamidase activity associated with cobra venom acetylcholinesterase: biological significance.
Biochimie, 2009Co-Authors: Ramanna V. Rajesh, Paul G. Layer, Rathanam BoopathyAbstract:Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is Aryl Acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.
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The Aryl Acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain.
Biochimie, 2009Co-Authors: Ramanna V. Rajesh, Paul G. Layer, Loganathan Chitra, Rathanam BoopathyAbstract: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 muM and 0.1 muM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 muM. 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.
Paul G. Layer - One of the best experts on this subject based on the ideXlab platform.
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High Aryl Acylamidase activity associated with cobra venom acetylcholinesterase: biological significance.
Biochimie, 2009Co-Authors: Ramanna V. Rajesh, Paul G. Layer, Rathanam BoopathyAbstract:Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is Aryl Acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.
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The Aryl Acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain.
Biochimie, 2009Co-Authors: Ramanna V. Rajesh, Paul G. Layer, Loganathan Chitra, Rathanam BoopathyAbstract: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 muM and 0.1 muM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 muM. 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.
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166 PUBLICATIONS 3,729 CITATIONS SEE PROFILE
2009Co-Authors: Rajesh Ramanna Valmiki, Paul G. Layer, Boopathy RathanamAbstract:High Aryl Acylamidase activity associated wit
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166 PUBLICATIONS 3,729 CITATIONS SEE PROFILE
2009Co-Authors: Rajesh Ramanna Valmiki, Paul G. Layer, Chitra Loganathan, Boopathy RathanamAbstract:The Aryl Acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brai
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Human serum cholinesterase from liver pathological samples exhibit highly elevated Aryl Acylamidase activity.
Clinica chimica acta; international journal of clinical chemistry, 2007Co-Authors: Rathanam Boopathy, Sultan Darvesh, Ramanna Valmiki Rajesh, Paul G. LayerAbstract:Although aspartate aminotransferase (AST) and gamma-glutamyltransferase (gamma GT) enzymes are widely used as markers for liver disorders, the ubiquitous enzyme butyrylcholinesterase (BChE), synthesized in liver is also used as marker in the assessment of liver pathophysiology. This BChE enzyme in addition to its esterase activity has yet another enzymatic function designated as Aryl Acylamidase (AAA) activity. It is determined in in vitro based on the hydrolysis of the synthetic substrate o-nitroacetanilide. In the present study, human serum cholinesterase (BChE) activity was studied with respect to its AAA activity on the BChE protein (AAA(BChE)) in patients with liver disorders. AST and gamma GT values were taken into account in this study as known markers for liver disorders. Blood samples were grouped into 3 based on esterase activity associated with BChE protein. They are normal, low, and very low BChE activity but with markedly increased AST and gamma GT levels. These samples were tested for their respective AAA function. Association of AAA with BChE from samples was proved using BChE monoclonal antibody precipitation experiment. The absolute levels of AAA were increased as BChE activity decreased while deviating from normal samples and such deviation was directly proportional to the severity of the liver disorder. Differences between these groups became prominent after determining the ratios of AAA(BChE) to BChE activities. Samples showing very high AAA(BChE) to BChE ratio were also showing high to very high gamma GT values. These findings establish AAA(BChE) as an independently regulated enzymatic activity on BChE especially in liver disorders. Moreover, since neither the low esterase activity of BChE by itself nor increased levels of AST/gamma GT are sufficient pathological indicators, this pilot study merits replication with large sample numbers.
Robert E Hoagland - One of the best experts on this subject based on the ideXlab platform.
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characterization of Aryl Acylamidase activity from propanil resistant barnyardgrass echinochloa crus galli l beauv
Weed Biology and Management, 2006Co-Authors: Kangetsu Hirase, Robert E HoaglandAbstract:The enzyme, Aryl Acylamidase, was characterized in propanil-susceptible and propanil-resistant barnyardgrass with respect to kinetic parameters, the effects of inhibitors, and the levels of activity in dark- and light-grown tissues. The enzyme reaction in the resistant tissue preparation proceeded linearly with time over a 5 h time course, while activity in the susceptible tissue preparation was 2- to 4-fold lower and the activity tended to decrease after 2 h. The apparent Km values were 62.1 mmol L −1 and 3.1 mmol L −1 for the enzyme activity in the susceptible and resistant tissue preparations, respectively. Two herbicides (anilofos and piperophos), previously shown to synergize propanil injury against the resistant biotype, were found to be potent inhibitors of the in vitro Aryl Acylamidase activity.
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The Rote of Plant and Microbial Hydrolytic Enzymes in Pesticide Metabolism
2006Co-Authors: Robert E Hoagland, Robert M. ZablotowiczAbstract:esters with carbonyl, phosphoryl, and thionyl linkages, are subject to enzymatic hydrolysis. Pesticide hydrolysis by esterases and amidases from plants and microorganlsms can serve as a deloxification or activation mechanism that can govern pesticide sel€ctivity or resistance, and initiate or determine the rate of pesticide biodegradation in the environment. Substrate specificity of esterases and amidases varies dramatically among species and biotypes of plants and microorganisms. The constitutive or induJible nature of these enzymes, as well as production of isozymes, is also important in the expression of these mechanisms' For example,'increased Aryl Acylamidase activity has been reported as the mechanism of evolved resistance to the hedicide Propanil in two Echinochloq weed species fiunglerice, E. colona (L.) Link; barnyardgrass, E. crus-Balli (L.) Beauv.l. Other Acylamidases, such as a linuron-inducible enzyme produced by Bacillus sphaericus' have broad substrate specificities including action on acylanilide' phenylcarbamate, and substituted phenylurea herbicides. Many rnicrobial hydrolytic enzymes are exfiacellular, thus hydrolysis can occur without uptake. Advances in molecular biology have led to an increased understanding of hydrolytic enzyme actiYe sites, especially those conferring selective specificity' This knowledge will create opportunities for engineering novel resistance mechanisms in plants and biosynthetic and/or degradative enzymes
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metabolically based resistance to the herbicide propanil in echinochloa species
Weed Science, 2004Co-Authors: Robert E Hoagland, F Carey, Jason K Norsworthy, R.e. TalbertAbstract:Abstract Propanil is an acylanilide herbicide introduced in the early 1960s to control dicotyledonous weeds and grasses, including Echinochloa species in cultivated rice. Since then, propanil has been used extensively in rice production in the United States and in several other countries. Propanil is an inhibitor of photosystem II, but rice is tolerant to propanil because of the presence of a high level of Aryl Acylamidase that catalytically degrades the compound to nonphytotoxic products, i.e., 3,4-dichloroaniline and propionic acid. About 10 yr ago, biotypes of barnyardgrass and junglerice were discovered to be resistant to propanil. The resistance mechanism of these two biotypes has been shown to be elevated levels of Aryl Acylamidase activity. Various strategies to combat propanil resistance and to more fully understand the biochemistry involved in this resistance have been investigated. These include studies on the interactions of herbicides and other chemicals with propanil, rotation of rice with ot...
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2-Nitroacetanilide as substrate for determination of Aryl Acylamidase activity in soils
Soil Biology and Biochemistry, 1998Co-Authors: Robert M. Zablotowicz, Robert E Hoagland, S.c. WagnerAbstract:Abstract A direct colorimetric assay for measuring Aryl Acylamidase activity in soils is described using 2-nitroacetanilide (2-NAA) as substrate. The assay is based upon an increase in absorption (410 nm) due to formation of the chromogenic hydrolysis product 2-nitroaniline, after incubation of soil with 2-NAA (2.0 m m ) in phosphate buffer (pH 8.0). Due to low specific activities (5–53 nmol g −1 soil h −1 ) incubations of 20–24 h are required. 2-NAA hydrolytic activity in soil was dependent on substrate concentration, with apparent K m values of 180 to 920 μ m for the soils examined. 2-NAA Aryl Acylamidase activity was linear for a 35 h incubation. The activity was reduced by 71 to 85% in the presence of carbAryl (100 μ m ), an Aryl Acylamidase inhibitor. Aryl Acylamidase activity was increased from 1.31 to 2.78-fold in no-till (NT) soils compared to conventional-till (CT) soils, corresponding with increased organic matter accumulation and microbial activity in the four (NT) soils studied.
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resistance mechanism of propanil resistant barnyardgrass ii in vivo metabolism of the propanil molecule
Pesticide Science, 1997Co-Authors: Frank V Carey, Robert E Hoagland, R.e. TalbertAbstract:Propanil-resistant barnyardgrass populations, previously verified in Arkansas rice fields and in greenhouse tests, were examined in the laboratory to ascertain if the resistance mechanism in this weed biotype was herbicide metabolism. Propanil-resistant barnyardgrass was controlled >95% in the greenhouse when carbAryl (an Aryl Acylamidase inhibitor) was applied two days prior to propanil. Laboratory studies with 14 C-radiolabelled propanil indicated that the herbicide was hydrolysed in propanil-resistant barnyardgrass and rice to form 3,4-dichloroaniline, but no detectable hydrolysis occurred in susceptible barnyardgrass. Two additional polar metabolites were detected in propanil-resistant barnyardgrass and rice and tentatively identified by thin layer chromatography. Overall, metabolites in the resistant barnyardgrass had R f values similar to those in rice, indicating similar metabolism for both species. These data, coupled with data from a previous report on the resistant biotype showing no differential absorption/translocation or molecular modification of the herbicide binding site in the resistant biotype, indicate that the resistance mechanism is metabolic degradation of propanil.
Ramanna V. Rajesh - One of the best experts on this subject based on the ideXlab platform.
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High Aryl Acylamidase activity associated with cobra venom acetylcholinesterase: biological significance.
Biochimie, 2009Co-Authors: Ramanna V. Rajesh, Paul G. Layer, Rathanam BoopathyAbstract:Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is Aryl Acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.
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The Aryl Acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain.
Biochimie, 2009Co-Authors: Ramanna V. Rajesh, Paul G. Layer, Loganathan Chitra, Rathanam BoopathyAbstract: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 muM and 0.1 muM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 muM. 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.
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Human serum cholinesterase from liver pathological samples exhibit highly elevated Aryl Acylamidase activity.
Clinica Chimica Acta, 2007Co-Authors: Rathanam Boopathy, Ramanna V. Rajesh, Darvesh, Paul G. LayerAbstract:Abstract Background Although aspartate aminotransferase (AST) and γ-glutamyltransferase (γGT) enzymes are widely used as markers for liver disorders, the ubiquitous enzyme butyrylcholinesterase (BChE), synthesized in liver is also used as marker in the assessment of liver pathophysiology. This BChE enzyme in addition to its esterase activity has yet another enzymatic function designated as Aryl Acylamidase (AAA) activity. It is determined in in vitro based on the hydrolysis of the synthetic substrate o -nitroacetanilide. In the present study, human serum cholinesterase (BChE) activity was studied with respect to its AAA activity on the BChE protein (AAA BChE ) in patients with liver disorders. AST and γGT values were taken into account in this study as known markers for liver disorders. Methods Blood samples were grouped into 3 based on esterase activity associated with BChE protein. They are normal, low, and very low BChE activity but with markedly increased AST and γGT levels. These samples were tested for their respective AAA function. Association of AAA with BChE from samples was proved using BChE monoclonal antibody precipitation experiment. Results The absolute levels of AAA were increased as BChE activity decreased while deviating from normal samples and such deviation was directly proportional to the severity of the liver disorder. Differences between these groups became prominent after determining the ratios of AAA BChE to BChE activities. Samples showing very high AAA BChE to BChE ratio were also showing high to very high γGT values. Conclusions These findings establish AAA BChE as an independently regulated enzymatic activity on BChE especially in liver disorders. Moreover, since neither the low esterase activity of BChE by itself nor increased levels of AST/γGT are sufficient pathological indicators, this pilot study merits replication with large sample numbers.
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Identification of serotonin-sensitive Aryl Acylamidase activity with cobra venom acetylcholinesterase
Indian journal of biochemistry & biophysics, 2003Co-Authors: Ramanna V. Rajesh, A S Balasubramanian, Rathanam BoopathyAbstract: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 antibody and showed the same heat denaturation profile at 40 degrees C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and Aryl Acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive Aryl Acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.
Shooichi Matsunaka - One of the best experts on this subject based on the ideXlab platform.
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Purification and properties of propanil hydrolase in Pseudomonas pickettii
Pesticide Biochemistry and Physiology, 1991Co-Authors: Kangetsu Hirase, Shooichi MatsunakaAbstract:Abstract Rice is resistant to propanil by virtue of an Aryl Acylamidase I enzyme which carries out detoxification of the herbicide. In order to obtain other crop plants resistant to propanil a bacterial source cally to transform plants, thus conferring herbicide resistance. For the primary stage, the purification and properties of a propanil hydrolase, isolated from a soil bacteria, Pseudomonas pickettii , was carried out. This enzyme was purified to homogeneity by ammonium sulfate fractionation, hydrophobic interaction chromatography, ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. The relative molecular weight was estimated to be 102,000 by HPLC equipped with gel filtration column. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 49,000. These results indicate that the propanil hydrolase from P. pickettii is a homodimer with subunit molecular weight of 49,000. The purified enzyme was stable for 42 days at 4°C. Amino acid composition of this enzyme was also determined.
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Physiological role of the propanil hydrolyzing enzyme (Aryl Acylamidase I) in rice plants
Pesticide Biochemistry and Physiology, 1991Co-Authors: Kangetsu Hirase, Shooichi MatsunakaAbstract:Abstract With the view of studying the physiological role of the propanil hydrolyzing enzyme (Aryl Acylamidase I), a comparison was made between the propanil susceptible rice mutant, which lacks the enzyme activity, and normal rice. Also, the properties of rice treated with a carbamate insecticide, a strong Aryl Acylamidase I inhibitor, were examined. The growth of the rice mutant was repressed approximately twice as much as that of the normal one by ammonium sulfate and urea. The mutant rice accumulated twice as much free ammonia as the normal one in the shoots under a high nitrogen supply. m-Tolyl methylcarbamate (MTMC) was used as the inhibitor of the enzyme. The growth of normal rice treated with MTMC was repressed 20% by ammonium sulfate but that of untreated rice was not affected at all. The propanil susceptible rice mutant, which lacks the activity of Aryl Acylamidase I and has higher susceptibility to ammonium sulfate by nature, showed no changes after MTMC treatment. The rice mutant is also more susceptible to a microb-originated herbicide bialaphos, l -2-amino-4-[(hydroxy)(methyl)phosphinoyl]butyryl- l -alanyl-alanine, of which the mode of action is assumed to be ammonia accumulation, than the normal one. The mutant was controlled 50% by the foliar application of 40 ppm bialaphos but normal rice was not affected at all at this concentration. Furthermore, twice as much free asparagine was found in the mutant rice than in the normal rice. Accordingly, it is suspected that Aryl Acylamidase I plays an important role in the normal nitrogen metabolism relating asparagine in rice plants.
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Correlation of propanil hydrolyzing enzyme activity with leaf morphology in wild rices of genome CCDD
Pesticide Biochemistry and Physiology, 1991Co-Authors: Chen Jian Jun, Shooichi MatsunakaAbstract:Abstract The propanil hydrolyzing enzyme, Aryl Acylamidase I (AAI) (Arylacylamine amidohydrolase, EC 3.5.1.13), was highly correlated ( r = −0.83) with leaf width in three species of genus Oryza with genome CCDD. The specific activity of AAI was lower in the leaves of wide-leafed plants and this was well-reflected in propanil phytotoxicity in those plants. There were no significant differences between conjugation of 3,4-dichloroaniline or the presence of AAI inhibitors in the crude enzyme solutions from the narrow-leafed and wide-leafed strains. The same relationship between AAI activity and leaf width was observed in interspecific F 1 hybrids involving genome CCDD. In those F 1 hybrids the wide- and narrow-leafed strains showed comparable AAI activity per leaf of equal length. It was concluded that the concentration of the enzyme in the CCDD plants was diluted by plant bulk in the wide-leafed strains and the correlation appeared to be the indirect effect of genes altering plant morphology, especially leaf area. The significance of the correlations is discussed in relation to propanil resistance and plant phylogenetics.
