Acetylcholinesterase is orientated facing the cytoplasmic side in membranes derived from sarcoplasmic reticulum.

(1) Microsomal membranes from white rabbit muscle enriched in sarcoplasmic reticulum (SR) were used to investigate the preferential localization of acetylcholinesterase (AChE) in these membranes. (2) Integrity and orientation of the vesicles was assessed by measuring the inulin-inaccessible space of the vesicles and its calcium-loading capacity. (3) Treatment of the membranes with diisopropyl phosphorofluoridate (DFP), an irreversible inhibitor which is free soluble in lipid, produced an almost complete inactivation of AChE. The inhibition was prevented in assays performed with the non-permeant reversible inhibitor BW 284c51 (BW). (4) Similar results were obtained if echothiophate iodide (ECHO), an irreversible and poorly permeant inhibitor, instead of DFP was used. (5) Sedimentation profiles of enzyme solubilized with Triton X-100 from membranes inhibited by DFP after protection with BW showed a minor reduction in the relative proportion of a 4.5 S (G1) form. (6) Treatment of intact or saponin-permeabilized membranes with concanavalin A (ConA) produced enzyme-lectin complexes. In both cases, most of the enzyme was recovered in the sedimented complexes after centrifugation of the Triton-solubilized membranes. (7) Incubation of intact membranes with the antibody AE1 led to the formation of immuno complexes. Sedimentation analyses of the molecular forms of AChE revealed a shift in the sedimentation coefficients, whether the antibody was added before or after solubilization of the enzyme. (8) These results firmly establish an external localization of AChE in SR, most of the protein backbone facing the cytoplasmic side of the membrane.

Amphiphilic and hydrophilic molecular forms of acetylcholinesterase in membranes derived from sarcoplasmic reticulum of skeletal muscle.

Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.

Interactions between lectins and acetylcholinesterase from the sarcotubular system of skeletal muscle.

The oligosaccharide chains attached to the multiple forms of acetylcholinesterase (AChE) in a fraction enriched in membranes of the sarcoplasmic reticulum and T-tubules of rabbit skeletal muscle were studied using lectins. By sequential extraction of the membranes with Triton X-100, two preparations of soluble enzyme were obtained, S(1) and S(2). Monomeric, tetrameric and asymmetric forms of AChE are present, and all of these bind to concanavalin A, yielding heavy aggregates with no loss of enzyme activity. The interaction of AChE with lectins suggests that the carbohydrate residues are N-linked to the protein backbone. The extent of the association of isolated AChE forms with wheatgerm and Ricinus communis agglutinins was variable, but grew as the molecular complexity increased. The interaction of monomeric AChE, in S(1) and S(2), with concanavalin A and Lens culinaris agglutinin suggests heterogeneity in the oligosaccharide moieties of this single form. Antibodies of the HNK-1 anti-carbohydrate type show no reaction with any of the AChE forms. Sialylation also appeared to be absent. The results overall indicate that some monomeric forms of AChE are fucosylated in the Golgi system to become precursors of tetrameric and asymmetric components of this enzyme in muscle.