ABSTRACT
In bovine brain cortex cytoplasm we have identified a soluble protein (L-protein) of M(r) approximately 90 kDa interacting with polyclonal antibodies to alpha-latrotoxin. The L-protein forms potential-dependent and cation-selective ion channels in BLM, which are blocked by Cd2+. The fusogenic activity of the L-protein was demonstrated on liposomes. We have arrived at the conclusion that the action mechanisms of the L-protein and alpha-latrotoxin are similar.
Subject(s)
Cerebral Cortex/chemistry , Nerve Tissue Proteins/chemistry , Spider Venoms/chemistry , Animals , Cattle , Lipid Bilayers/chemistry , Liposomes/chemistry , Membrane Potentials , Molecular Weight , Nerve Tissue Proteins/isolation & purification , SolubilityABSTRACT
Phosphorylation of the reconstructed TTX-sensitive cytosolic protein of the bovine brain has been studied. Some properties of the protein are similar to those of the membrane potential-dependent sodium channel. It is shown that the influence of phosphorylation by protein kinase A on the reconstructed channel greatly depends on the mode of reconstruction. Phosphorylation fo reconstructed channels in the open state leads to their closing. Preliminary phosphorylation of channel-forming protein results in a considerable increase of the activation effect of veratrine and scorpion toxin.
Subject(s)
Membranes, Artificial , Protein Kinases/metabolism , Sodium Channels/physiology , Animals , Cattle , Membrane Potentials/physiology , Nerve Tissue Proteins/metabolism , Phosphorylation , Scorpion Venoms/pharmacology , Sodium Channels/drug effects , Tetrodotoxin/pharmacology , Veratrine/pharmacologyABSTRACT
Biochemical events leading to the formation of mature membrane-associated sodium channel proteins are not completely understood. We have recently purified a protein from the cytoplasm of brain cells, which is able to become incorporated into liposomes and induce neurotoxin-dependent sodium permeability. Here we report data on a monoclonal antibody derived against this protein. This antibody crossreacts with cell membrane preparations. The antibody binding to viable neuroblastoma cells is inhibited by veratrine, indicating that membrane molecules antigenically related to the cytoplasmic protein may also be related to the voltage-gated sodium channel.
Subject(s)
Antibodies, Monoclonal , Brain/metabolism , Ion Channel Gating , Nerve Tissue Proteins/physiology , Sodium Channels/physiology , Tetrodotoxin/pharmacology , Animals , Antibodies, Monoclonal/immunology , Cytoplasm/metabolism , Drug Resistance , Electrophysiology , Female , Immunization , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins/immunology , Neurotoxins/pharmacology , Tumor Cells, Cultured , Veratrine/pharmacologySubject(s)
Brain/metabolism , Ion Channels/metabolism , Sodium Channels , Tetrodotoxin/metabolism , Animals , Carrier Proteins/metabolism , Cattle , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Immune Sera , Liposomes/metabolism , Membrane Proteins/isolation & purification , RabbitsABSTRACT
Reconstruction of membrane proteins obtained from the Sepharose-rabbit immunoglobulins to bovine brain TTX-sensitive cytoplasmic protein column has been studied. The detergent method for studying potential-dependent sodium channel has been used in our modification. The results obtained indicate that cytoplasmic TTX-sensitive proteins are close to those of the membrane sodium channel.
Subject(s)
Carrier Proteins/immunology , Membrane Proteins/immunology , Neurons/physiology , Animals , Brain/metabolism , Carrier Proteins/isolation & purification , Cattle , Electrophoresis, Polyacrylamide Gel , Ion Channels , Membrane Proteins/isolation & purification , Sodium/metabolism , Sodium Channels/physiologyABSTRACT
Hypothetical antigenic similarities between nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins have been studied. Indirect ELISA binding assay combined with inhibition assay has been used. The results obtained indicate that cytoplasmic tetrodotoxin-sensitive proteins do share antigenic determinants with nerve cell membrane structures. This is consistent with the speculation that cytoplasmic tetrodotoxin-sensitive proteins are relatives of membrane sodium channels.
Subject(s)
Epitopes/analysis , Membrane Proteins/immunology , Nerve Tissue Proteins/immunology , Neurons/immunology , Tetrodotoxin/pharmacology , Animals , Brain/immunology , Cattle , Cell Line , Cell Membrane/immunology , Cytoplasm/immunology , Mice , Neuroblastoma , Rats , Synaptosomes/immunologyABSTRACT
Highly purified protein inducing tetrodotoxin-dependent Na fluxes in liposomal membrane was obtained from the cytoplasmic fraction of the bovine brain. The protein was purified by anion-exchange chromatography on DEAE-Servacell and wheat germ agglutinin sepharose (WGA) followed by gel filtration on sepharose 4B. It is a high-molecular weight acidic glycoprotein; during denaturation under reducing conditions it forms 55 kD subunits. It is suggested that the tetrodotoxin-sensitive protein could be a soluble intracellular precursor of the voltage-dependent sodium channels.
Subject(s)
Brain Chemistry , Glycoproteins/isolation & purification , Ion Channels/analysis , Nerve Tissue Proteins/isolation & purification , Sodium/metabolism , Tetrodotoxin/pharmacology , Animals , Brain/drug effects , Cattle , Chemical Phenomena , Chemistry , Ion Channels/drug effectsABSTRACT
The peculiarities of interaction of TTX-sensitive soluble cytoplasmic brain fraction proteins with liposomes were studied. It was shown that transmembrane orientated insertion of TTX-sensitive proteins into liposomes depends on chemical composition of their lipids.
