Cellular localization of the molecular forms of acetylcholinesterase in cultured embryonic rat myotubes.

Three different methods were used to quantitate the external and intracellular acetylcholinesterase (AChE) in cultured embryonic rat myotubes. The results from these methods were in excellent agreement and showed that one-fourth of the AChE is external and three-fourths is intracellular in these cells. The molecular forms of AChE in the intracellular and external compartments then were analyzed. To isolate intracellular AChE, external AChE was inactivated irreversibly by treating myotubes at 2°C with 10 PM methanesulfonyl fluoride and 100 PM decamethonium for 1 hr. To isolate external AChE, myotubes at 2°C were first exposed to 1.0 PM echothiophate for 15 min to protect (diethylphosphorylate) external AChE and then treated with 1.0 mM methanesulfonyl fluoride for 30 min at 37°C to inactivate intracellular AChE irreversibly. Control myotubes and myotubes in which the external or intracellular enzyme had been isolated were extracted sequentially to separate globular, asymmetric, and nonextractable AChE. Individual globular and asymmetric forms then were analyzed by velocity sedimentation on sucrose gradients. The fractions in which external enzyme had been isolated as diethylphosphorylated AChE were reactivated with l-methyl2-hydroxyiminomethylpyridinium prior to analysis. Our data indicate that intracellular and external AChE have different compositions. Intracellular AChE is 79% globular forms (20% 10 S and 59% 4 S), 17% asymmetric forms (6% 16 S, 5% 12.5 S, and 7% other), and 4% nonextractable enzyme. External AChE is 55% globular forms (26% 10 S and 29% 4 S), 32% asymmetric forms (12% 16 S, 10% 12.5 S, and 10% other), and 13% nonextractable enzyme; therefore external enzyme is composed of relatively more 10 S, 16 S, 12.5 S, and nonextractable AChE and relatively less 4 S AChE. The most striking finding to emerge from this study is that the globular and asymmetric forms are all predominantly intracellular in cultured embryonic rat myotubes. Our results indicate that 85% of the 4 S, 69% of the 10 S, 59% of the 16 S, and 56% of the 12.5 S forms are intracellular. These results support the hypothesis that the 10 S and asymmetric forms of AChE are assembled intracellularly from 4 S precursors. The enzyme acetylcholinesterase (AChE) occurs in a isolated from the electric organ of the eel Electrophorus number of molecular forms. Three of these forms are electricus. The three asymmetric forms are designated globular proteins and three are asymmetric. The globular Ad, As, and Alz (Bon et al., 1979) and consist of one, two, forms are monomers, dimers, and tetramers of the basic or three tetrameric assemblies of identical catalytic subcatalytic subunit designated G1, Gz, and Gd (Bon et al., units attached to an elongated collagen-like tail (Bon et 1979; Vigny et al., 1979). The structure of the asymmetric al., 1976; Rosenberry and Richardson, 1977; Anglister forms has been analyzed most carefully using AChE and Silman, 1978). ’ We wish to thank Dr. T. L. Rosenberry for his advice throughout In adult rat skeletal muscle, there are major forms of this study and for his comments on the manuscript. This investigation AChE that sediment at 16 S (A12), 10 S (G4), and 4 S was supported by National Institutes of Health Grants NS 15219 and (G1), appreciable enzyme that sediments at 12.5 S (&), 16577, by National Institutes of Health Medical Scientist Training and a minor form that sediments at 6.5 S (Gz) (Hall, Grant GM-07250 (S. K. B.), and by a grant from the Muscular Dystro1973; Vigny et al., 1976; Carson et al., 1979; Bon et al., phy Association. 1979; Younkin et al., 1982). In a recent study of the adult ‘To whom correspondence should be addressed at Department of rat diaphragm, we discovered (Younkin et al., 1982) that Pharmacology, Case Western Reserve University School of Medicine, 30% of the asymmetric forms and 34% of the 10 S AChE Cleveland, OH 44106. are intracellular. These findings led us to propose that