A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels

Abstract

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. -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. A novel method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels has been developed.

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

-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.

Background

].

].

]. The natural substrate or the precise physiological role of the AAA activity on ChEs is not known.

Studies on the tissue specific and developmental regulations/alterations of AAA on ChEs have not been attempted so far partly due to the lower specific activity of the AAA activity on ChEs and mainly due to the absence of a specific method to visualise this activity. In the present paper, a method to visualise the AAA activity on ChEs in polyacrylamide gels using human serum BChE and electric eel AChE as models is described.

Results and discussion

].

Scheme representing the principle of staining of the AAA actitvity.

-nitroaniline to form the diazonium compound. For optimal detection of the AAA activity, a concentration of 0.1 % (w/v) sodium nitrite and 1 N HCl were found to be required. Similarly, studies with varying concentration of NED indicated that the maximum intensity of colour developed was with 0.75 % (w/v) of the colouring reagent.

, it is also clear that, to visualise a recognisable AAA activity band in the gel, at least 0.07 U of the enzyme is required.

Differential staining for the AAA activities on ChEs from eel AChE and human serum BChE. Electrophoresis was performed on 3.5% native polyacrylamide gel slabs under non-denaturing conditions as detailed in 'Materials and methods'. All lanes were loaded with 1 U each of AAA on eel AChE and AAA on human serum BChE . After electrophoresis, 'A' was incubated in substrate solution without any inhibitor; 'B' was incubated in substrate solution containing 100 μM of iso-OMPA; and 'C' was incubated in substrate solution containing 100 μM of BW 284c51. After incubation for 40 min, the gels were stained for the AAA activity. The upper and lower arrows denote the AAA activity due to BChE and AChE, respectively.

Effect of enzyme concentration (in terms of AAA activity) on the intensity of staining. Electrophoresis was performed on 3.5% native polyacrylamide gel as described under 'Materials and methods'. Lanes 1-5, respectively, corresponds to 0.07 U, 0.14 U, 0.21 U, 0.28 U and 0.35 U of AAA activity on BChE. After electrophoresis, the gel was stained for the AAA activity as given under 'Materials and Methods'.

).

).

α-Naphthol and β-naphthol were also tried as colouring agents instead of NED. In alkaline conditions, α-naphthol produced a red coloured band, while β-naphthol produced an orange coloured band (Figure not given). When compared to the colour developed with NED, the colour developed with either of the naphthols was faint and diffused. Moreover, the fixing of the stain in the gels was also difficult.

-nitroacetanilide as substrate can be visualised by the method described above. This method would also allow detection of such activities in crude tissue extracts, however, a minimum of 0.07 U of the enzyme has to be loaded per well to clearly visualise the activity in gels, which is the limit of detectability of this method.

Conclusion

.

Materials

-Nitroacetanilide was prepared as described in reference 16.

Enzymes

]. Electric eel AChE (product No. C 3389) was procured from Sigma Chemical Co., St. Louis, USA.

-nitroaniline in 1 hr under the standard assay conditions.

Staining procedure

]. After electrophoresis, the gels were stained for the AAA activity as follows. The gels were incubated in the substrate solution (10 mM ONAA in 0.1 M potassium phosphate buffer, pH 7.0) for 45 min at 40°C. The gels were transferred from the substrate solution to ice-cold staining solution of 1 N HCl containing 0.1% freshly prepared sodium nitrite and agitated briefly. Then 150 mg (final concentration 0.75% w/v) of N-(1-napthyl)ethylenediamine was added and the gels were gently agitated until sufficient intensity of the characteristic purple colour bands developed. The gels were then transferred to 0.3 M trichloroacetic acid (TCA) and kept for 30 min at 4°C. The gels were finally washed with 0.1 M Tris-HCl buffer, pH 8.6 and stored in the same buffer in a refrigerator. Differential staining for AChE AAA activity was performed by incubating the gels in the substrate solution containing 100 μM iso-OMPA to inhibit the AAA on BChE. Alternatively, in order to visualise BChE AAA activity, gels were incubated in substrate solution containing 100 μM BW 284c51 to inhibit the AAA on AChE.

Acknowledgements

The support by Defence Research & Development Establishment (DRDE), Gwalior, India (Project ID No. TC/05414/DRDE/Project-DRDE-149, dated 23 June, 1998) and the award of Senior Research Fellowship to L.J. by the Council of Scientific and Industrial Research, India, and DRDE are gratefully acknowledged.

Abbreviations

-nitrooacetanilide; TCA, trichloroaceticacid.