Abstract
Heparin is capable of solubilizing a subset of collagen-tailed (A12) acetylcholinesterase (AChE) molecules from skeletal basal lamina (Rossi, S. G., and Rotundo, R. L. (1993) J. Biol. Chem. 268, 19152-19159). In the present study, we used tissue-cultured quail myotubes to show that, like adult fibers, neither heparin- nor high salt-containing buffers detached AChE molecules from cell-surface clusters. Prelabeling clustered AChE molecules with anti-AchE monoclonal antibody 1A2 followed by incubation in heparin-containing medium showed that there was no reduction in the number or size of preexisting AChE clusters. In contrast, incubation of myotubes with culture medium containing heparin for up to 4 days reversibly blocked the accumulation of new cell-surface AChE molecules without affecting the rate of AChE synthesis or assembly. Newly synthesized A12 AChE becomes tightly attached to the extracellular matrix following externalization. However, in the presence of heparin, blocking the initial interactions between A12 AChE and the extracellular matrix results in release of AChE into the medium with a t1/2 of approximately 3 h. Together, these results suggest that once A12 AChE is localized on the cell surface, initially attached via electrostatic interactions, additional factors or events are responsible for its selective and more permanent retention on the basal lamina.
Highlights
Catalytic subunits are locally translated and assembled around the nuclei encoding their transcripts (Rotundo, 1990), and the newly synthesized AChE oligomers are selectively localized to regions of the cell surface over the nucleus of origin (Rossi and Rotundo, 1992)
To determine whether high salt- or heparin-containing buffers could detach clustered AChE molecules from the extracellular matrix, 7-day-old quail muscle cultures were incubated for 1 h in either Low salt extraction buffer (LSB) or high salt extraction buffer (HSB) with or without 0.5 mg/ml heparin
Its mechanism of attachment to the synaptic basal lamina is generally thought to be through electrostatic interactions with glycosaminoglycans such as heparan sulfate proteoglycan or dermatan sulfate proteoglycan (Bon et al, 1978; Brandan and Inestrosa, 1987; Perez-Tur et al, 1991b; Melo and Brandan, 1993)
Summary
Preparation of Tissue-cultured Muscle—Pectoral muscle cells from 10-day-old quail embryos were grown on scratched collagen-coated coverslips (25-mm diameter) in Eagle’s minimal essential medium supplemented with 2% chick embryo extract, 10% horse serum, and 50 g/ml gentamicin (EMEM 210) as described previously (Rossi and Rotundo, 1992). 20-l aliquots from each culture were removed and mixed with 1.5 ml of 50 mM glycine HCl buffer (pH 2.5) with 2 M NaCl in scintillation vials, and the labeled acetate was counted by addition of 5 ml of ACS scintillation fluid containing 20% 1-butanol (v/v). Under these assay conditions, the assay was linear for Ͼ1 h. AChE oligomeric forms expressed on the cell surface were analyzed by velocity sedimentation following protection with the water-soluble reversible AChE inhibitor BW284c51 and irreversible inactivation of the intracellular AChE with diisopropyl fluorophosphate (DFP) as described previously (Rotundo, 1984b). The results are expressed as clusters per myotube nuclei (mean Ϯ S.E.)
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