Studies on acetylcholinesterase and cell wall proteins in Phaseolus vulgaris L.
Creators: name: Mansfield, Donald Holmes
Publisher: name: University of British Columbia
Year: 2010

description: Acetylcholinesterase (AchE) activity r was identified in roots and hypocotyls of etiolated Phaseolus vulgaris L. by means of a colorimetric assay which included the cholinesterase inhibitor neostigmine as a control. An inhibitor of this activity was observed in tissue homogenates but was removed by dialysis. Greater than 95% of the activity in the hypocotyl was localized in the cell walls. The enzyme was extracted from the buffer-insoluble residue of roots with 5% (NH₄)₂SO₄ and purified by (NH₄)₂SO₄ precipitation, gel filtration on Sepharose 6B and chromatography on N-methylacridinium-Sepharose 4B. Purified preparations had a specific activity of 210 ± 20 units mg⁻¹ protein and contained one active protein and one major inactive protein as determined by polyaerylamide gel electrophoresis and thin layer isoelectric focusing. The AchE had at least 3-fold greater activity against acetylthiocholine than butyl- or propionylthiocholine. The K[sub m] of AchE for acetylthiocholine was 56 μM. The enzyme was stimulated by choline (0.5-50 mM) and totally inhibited by diisopropylfluorophosphate (DIFP, 10⁻⁴ M) and decamethonium (60 mM.). The catalytic center activity determined by DIFP titration was 197 ± 5 mol substrate min⁻¹ mol⁻¹ active center. The isoelectric point of AchE was 5.3 ± 0.1, the sedimentation coefficient (S[sub 20,w]) was 4.2 ± 0.1 S, and the Stokes radius was 4.00 nm. The mol. wt. calculated from sedimentation and gel filtration data was 76000 ± 2000. The mol. wt. determined by SDS-gel electrophoresis was 77000 ± 2000. Subunit mol. wts. of 61000 ± 2000 (2 x 30000) and 26000 ± 2000 were observed. The enzyme had a frictional ratio (f/fo) of 1.37. A theoretical model of the quaternary structure of AchE was presented. Multiple forms of AchE activity were observed following gel filtration in low ionic strength and ion exchange chromatography of preparations having low specific activity. It was suggested that an ionic strength dependent equilibrium existed between aggregates of the 77000 mol. wt. species. Properties of the bean root AchE were compared with the AchEs from eel and other animal tissues. Though large differences existed, in catalytic center activities, substrate hydrolysis rates, and behavior on N-methylacridinium-Sepharose 4B, the AchE from the different sources were similar in many respects. The specific activity of hypocotyl hook AchE was unaffected by exposure of etiolated seedlings to light or an ethylene source for 3 days. The specific activity of hook AchE increased after 3 days in 10⁻³ M gibberellin-treated plants and decreased after 4 days in 10⁻⁴ M-kinetin-treated plants. These results were interpreted in terms of a possible role of AchE in hypocotyl aging. In a second study, walls of etiolated P. vulgaris hypocotyls were treated with ³²P-DIFP under conditions which corrected for adsorption of the radioisotope, to determine the number of nucleophilic sites in cell walls from regions having different elongation rates. Alpha- chymotrypsin and serine were treated similarly to establish optimum conditions for diisopropylphosphorylation. The ³²P-phosphoserine content of partially hydrolyzed cell walls was determined. Cell walls from regions of active cell elongation contained 2.71 pmol ³²P-phosphoserine mg⁻¹ cell wall and those from regions in which cell elongation had terminated contained no significant ³²P-phosphoserine. From these results I concluded that the few nucleophilic sites present were, not glycosylation sites in the cell wall but rather were sites in active centers of enzymes which were bound to cell wall preparations.