Abstract
The cellular localization of the molecular forms of acetylcholinesterase was explored in chick sympathetic neurons and in mouse T28 cells (neuroblastoma X sympathetic ganglion cell hybrids) using the reversible, poorly lipid-soluble inhibitor of acetylcholinesterase, BW284C51, to protect cell surface activity while inactivating cytoplasmic activity with DFP, an irreversible, lipid-soluble inhibitor. Our results show protection of over 80% of the chick 11 S form and over 90% of the corresponding mouse 10 S form under these conditions, while over 90% of the chick 6.5 S and mouse 4 S forms are inhibited. The results suggest that the avian 11 S and mouse 10 S forms are predominantly or exclusively ectoenzymes while the respective 6.5 S and 4 S forms are confined to the cytoplasm.
Highlights
From the Departmentof Neuropathology, Harvard Medical School, Boston, Massachusetts 02115 a n d the Departmentof Pharmacology, New York University Medical Center, New York, New York 10016
T h e behavior, localization, and biochemistry of these forms have beenextensively studied in dithio(bis)dinitrobenzoic acid, and tetraisopropylpyrophosphoramide were from Sigma and pyridine-2-aldoxime methiodide from Ayerst Laboratories(New York, NY)
After detergent solubili- containing solutions were prepared within 5 min of use by dilution zation, these forms can beidentifiedbyenzymatic activity sedimenting in sucrose gradientswith coefficients of approximately 4 S, 6.5 S, 10 S,and 16 S in mammals ( 3,6-8) and 6.5 S, 11S, and 19.5 S in birds [3,9, 10].The 16 Sand corresponding 19.5 S forms have been found at the motor endplate(3, 6, from a 51 mM stock solution of DFP in propylene glycol
Summary
From the Departmentof Neuropathology, Harvard Medical School, Boston, Massachusetts 02115 a n d the Departmentof Pharmacology, New York University Medical Center, New York, New York 10016. After detergent solubili- containing solutions were prepared within 5 min of use by dilution zation, these forms can beidentifiedbyenzymatic activity sedimenting in sucrose gradientswith coefficients of approximately 4 S, 6.5 S, 10 S,and 16 S in mammals ( 3 ,6-8) and 6.5 S, 11S, and 19.5 S in birds [3,9, 10].The 16 Sand corresponding 19.5 S forms have been found at the motor endplate(3, 6, from a 51 mM stock solution of DFP in propylene glycol The latter was sequentially diluted1:10in PBS and then1:200in the appropriate solution. The final concentrations of DFP and propylene glycol were 2.5 x
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