Marmorata Electric Organ Research Articles

Dystrophin and dystrophin-related protein expression in Torpedo marmorata electric organ

The presence of different dystrophin-related protein forms was investigated in electric organ as compared to cardiac, white or red skeletal muscles from Torpedo marmorata. Two strategies were followed. First, we used specific C-terminal dystrophin and dystrophin-related protein monoclonal antibodies which we characterized in the present study. 400 kDa protein bands were detected in the tissues mentioned above with both specific types of antibodies. Second, we produced monoclonal antibodies raised against a dystrophin-enriched preparation from T. marmorata electric organ. Western blot and immunofluorescence analyses showed the tissue specificity of T. marmorata antibodies and allowed us to classify them as types I, II and III. Vessel walls and neuromuscular junctions were labeled with T. marmorata type II and III antibodies in human muscles (skeletal and smooth). Both approaches demonstrated that the T. marmorata electric organ contained different proteins related with dystrophin: a dystrophin form, a dystrophin-related protein form and a dystrophin-related protein isoform, homologous to the dystrophin-related protein present in muscle vessel walls and at the neuromuscular junctions of human tissues. The presence of dystrophin and dystrophin-related protein is finally discussed relative to their functions and organ specificities.

Asymmetric distribution of phospholipids in acetylcholine receptor-rich membranes from T. marmorata electric organ

1. 1. The distribution of phospholipids between the two leaflets of the lipid bilayer in acetylcholine receptor (AChR)-rich membranes from T. marmorata has been examined with two complementary techniques: chemical derivatization with the membrane-impermeable reagent trinitrobenzenesulphonate (TNBS) and B. cereus phospholipase C hydrolysis. 2. 2. AChR-membranes were reacted with TNBS at 0–4 and 37°C and the accessibility of their aminophospholipids was compared to that of rod outer segment and erythrocyte membranes. The results indicate that more of the total ethanolamine glycerophospholipid (EGP) than of the total phosphatidylserine (PS) is located in the outer monolayer. 3. 3. Nearly half the phospholipid content of AChR membranes is hydrolyzed by phospholipase C with a half-time of ca 1.6 min at 25°C. Consistent with the TNBS results, more of the total EGP than of the total PS is degraded. Beyond 3 min the reaction slows down, relatively smaller additional amounts of lipids are hydrolyzed, and all phospholipid classes are attacked to a similar extent, indicating that after half the lipid is removed all phospholipids become accessible to the enzyme. 4. 4. The results indicate that the outer leaflet of the bilayer is richer in ethanolamine and choline glycerophospholipids, whereas phosphatidylinositol, most of the sphingomyelin, and ca 65% of the PS are located on the inner leaflet.

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes.

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000-43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.

An ESR study of the postsynaptic membrane acetylcholinesterase of torpedo marmorata electric organ

The effect of the cholinergic activator, phenyltrimethylammonium, on the ESR spectra of spin-labeled membrane bound acetylcholinesterase was studied; a reduction of maximal hyperfine splitting of the anisotropic ESR spectrum by 2 G was observed. The influence of phenyltrimethylammonium was prevented by the two cholinergic blocking agents d-tubocurarine and alpha-cobratoxine. The present results indicate that the conformation change of the esteratic site of membrane acetylcholinesterase is triggered by the binding of phenyltrimethylammonium to the cholinoreceptor site.