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

Abstract

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-methyl-2-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 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 Vigny et al., 1979). The structure of the asymmetric al. Rosenberry and Richardson, 1977; Anglister forms has been analyzed most carefully using AChE and Silman, 1978).

Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

“…Incubations were carried out with 1 jiM echothiophate in phosphate-buffered saline (PBS) for 20 min at 4°Cto minimize pinocytosis (Brockman et al, 1982). After incubation with inhibitor, cells were rinsed 4 X 20 min each with PBS at 4°Cand sonicated in 0.05 M Tris buffer, pH 7.4, with 1% Triton X-100.…”

Asymmetric acetylcholinesterase is assembled in the Golgi apparatus.

“…The time from synthesis until appearance at the plasma membrane is approximately 2.5 hr in both the chicken and quail muscle cultures. More recently, wet and others (22)(23)(24) have shown that both asymmetric and globular forms of AcChoEase are found within the muscle cells, suggesting that their assembly is intracellular. Based upon these observations, we can begin to ask where and when in the cell are the several molecular forms of this glycoprotein assembled.…”

Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

“…This enzyme species is composed of three sets of four catalytic subunits disulfide bonded to a collagen-like tail and is thought to be the one present in muscle basal lamina (25)(26)(27). Numerous investigators have shown that a variety of types of muscle fibers grown in neuron-free primary cell culture and permitted to undergo spontaneous contraction are able to synthesize several forms of AcChoE including the A12 (28)(29)(30)(31). However, if contraction of rat muscle fibers, for example, is blocked by growing the fibers in the presence of tetrodotoxin (TTX), both the total amount of AcChoE and the percentage of AcChoE assembled as the A12 form are decreased severalfold (32)(33)(34).…”