Characterization of Calelectrin, a Ca2+-Binding Protein Isolated from the Electric Organ of Torpedo marmorata

We report a fast (less than 1 day) and efficient (2-3 mg protein/100 g tissue) isolation method for calelectrin, a protein of Mr 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca2+. Purified protein was used to investigate the nature of its interaction with membranes and with Ca2+. Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca2+-dependent and -specific manner with half-maximal binding between 3 and 7 microM free Ca2+. This binding is totally inhibited by 1 mM mercaptoethanol. It is also shown that calelectrin directly binds Ca2+ in solution by two techniques: at 1 and 10 microM Ca2+ it binds 45Ca2+ as measured by gel permeation chromatography, and it contains saturable Tb3+-binding sites that are Ca2+-displaceable. An investigation of the protein's endogenous fluorescence shows that although it contains both tryptophan and tyrosine, there is no change in the apparent quantum yield as a function of Ca2+. Ca2+-dependent hydrophobic affinity chromatography of the total soluble proteins from Torpedo electric organ shows that Torpedo calelectrin, like calmodulin and mammalian calelectrins, is specifically retained in the presence of Ca2+ and eluted by EGTA. Calelectrin also contains high-affinity sites for hydrophobic fluorescence probes such as N-phenyl-1-naphthylamine, 2-CP-toluidinylnaphthalene-6-sulfonic acid, and 1-anilinonaphthalene-8-sulfonic acid, which again unlike calmodulin, show no changes as a function of Ca2+. We conclude that calelectrin is a Ca2+-binding protein whose binding to the lipid moieties of membranes is regulated by physiological change in the Ca2+ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calelectrin self-aggregates and promotes membrane aggregation in the presence of calcium.

Calelectrin is a protein that can be purified to homogeneity from the cholinergically innervated electric organ of Torpedo marmorata where it is present in large amounts. It has been shown to bind to the membranes of the electric organ in a Ca2+-dependent and specific manner. Using the purified protein we now report that it is specifically self-aggregated by Ca2+ in micromolar concentrations but not by Mg2+ at much higher concentrations. Sr2+ is also completely inactive, while Ba2+ and the trivalent lanthanides Tb3+, Eu3 +, and La3+ can substitute for Ca2+. Calelectrin also greatly enhances the Ca2+-induced aggregation of isolated synaptic vesicle membranes from the cholinergic nerve terminals of T. marmorata and of chromaffin granule membranes from the bovine adrenal medulla. The potentiation of membrane aggregation is mainly due to the appearance of a fast aggregatory phase in the presence of calelectrin . It is saturable with respect to calelectrin and can be demonstrated at very low calelectrin concentrations, suggesting a specific calelectrin membrane-binding component. This component seems to be of lipid nature since the aggregation of total extracted lipids from Torpedo electric organ and from chromaffin granules could also be enhanced by calelectrin . The Ca2+-induced self-association of calelectrin and its aggregation enhancing effect may be of great importance to the structural organization of neural and secretory cells and the mechanism of exocytosis.     

Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo

Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.     

Isolation of mammalian calelectrins: a new class of ubiquitous Ca2+-regulated proteins

In a new approach to isolating proteins which participate in the Ca2+-dependent regulation of membrane traffic in animal cells, two new Ca2+-binding proteins (Mr 67 000 and 32 500) have been identified in and purified from bovine liver, brain, and adrenal medulla. These proteins specifically and reversibly bind to chromaffin granule membranes at low Ca2+ concentrations (half-maximal binding at 5.5 microM Ca2+) and greatly potentiate the Ca2+-induced aggregation of these membranes at higher concentrations (above 10 microM). In the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, the isolated proteins have Stokes radii of 3.40 nm (Mr 67 000) and 2.53 nm (Mr 32 500) as estimated by gel filtration and therefore occur as monomers. They are slightly acidic proteins with pI's of 5.85 and 5.60. In bovine tissues, both proteins and a third protein of Mr 35 000 cross-react immunologically with each other and with Torpedo calelectrin (Mr 34 000) and are therefore identified as mammalian calelectrins. In all tissues of Torpedo marmorata tested, only a single molecular mass form of calelectrin exists, whereas multiple forms of calelectrin exist in mammalian tissues, indicating gene duplication during evolution. We suggest that the evolutionary conservation and diversification, the high tissue concentrations, and the Ca2+-specific interactions of the calelectrins make them candidates for Ca2+-dependent regulators of membrane events in animal cells.     

Calelectrin, a Calcium-Dependent Membrane-Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla

Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.     

Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin.

Addition of Ca2+ to post-microsomal fractions of bovine adrenal or liver produced a sedimentable complex of membrane vesicles and cytoplasmic proteins. Proteins with apparent mol. wts. 70 000, 36 000 and 32 500 were solubilized from this complex by Ca2+ chelation. The 36 000 mol. wt. protein (p36) was immunoprecipitated by an antiserum specific for pp36, a major substrate for Rous sarcoma virus src-gene tyrosine kinase. This protein was present in many mesenchymal cells and associated with membrane cytoskeleton of bovine fibroblasts in a Ca2+-dependent manner. The 70 000 and 32 500 mol. wt. proteins were widely distributed in established cell lines, but were not clearly associated with cell organelles in tissue sections, nor retained in cytoskeleton preparations. On immunoblots p36 reacted strongly with antibodies produced against the electric fish protein Torpedo calelectrin and the similar Ca2+-binding properties and subunit mol. wts. of these proteins suggests that they might be functionally related. Since Torpedo calelectrin, p70, p36 and p32.5 were bound by lipid vesicles or microsomal membranes at micromolar free Ca2+ concentrations, regulated association with intrinsic membrane components may be involved in the functions of these widespread proteins.     

Ca2+- and phospholipid-binding properties of Torpedo electric organ calelectrin

The Ca2+-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ of Torpedo marmorata have been compared. Binding of H-calelectrin required Ca2+ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca2+ took place only when the Ca2+ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca2+.Mg2+, Na+ and K+ inhibited the Ca2+-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hydrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated "down-regulated" form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca2+, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca2+-regulated.     

Isolation from Cholinergic Synapses of a Protein That Binds to Membranes in a Calcium-Dependent Manner

A protein that binds to membranes in a calcium-dependent manner between calcium concentrations of 10(-5) and 10(-6) M has been isolated in large amounts (20 mg/kg tissue) from the entirely cholinergic electric organ of Torpedo marmorata. The protein bound reversibly to membrane fractions in a calcium-specific and saturable manner. The protein also bound to lipids isolated from Torpedo electric organ and to clathrin-coated vesicles prepared from pig brain. The protein bound to a Triton X-100-sensitive site. It had an apparent subunit molecular weight of 32,000 by polyacrylamide gel electrophoresis and of 35,900 by amino acid analysis; a broad isoelectric range of 4.8 to 5.5; and 27% of its amino acids after hydrolysis were observed to be aspartic and glutamic acids. Synaptosomes derived from electric organ were enriched in the protein which is probably localised within the nerve ending. It was localised in the synaptic region of the electric organ by means of immunofluorescence. In the electric lobe, discrete patches of fluorescence were seen within the cell bodies that innervate the electric organ. The protein may be involved in the recognition of membranes within the cholinergic neurone. Proteins with similar purification properties were found in all tissues investigated so far, and polypeptides of subunit molecular weight 32,000 were identified in bovine adrenal medulla and guinea pig brain synaptosomes.     

Isolation and characterization of two novel calcium-dependent phospholipid-binding proteins from bovine lung

Two calcium-dependent proteins of apparent Mr 32,000 and 34,000 were isolated from bovine lung. Approx. 70 mg/kg of each was obtained. Two-dimensional gel electrophoresis in the presence of 8 M urea showed their apparent p/values to be 5.1 and 5.0, respectively. Both proteins are related immunologically to calelectrin from Torpedo marmorata. They also have very similar amino acid compositions to calelectrin. Partial sequence information shows that both proteins contain the highly conserved sequence described for the annexins, a new family of calcium-dependent membrane-binding proteins. In common with other members of this family, the new proteins bind to acidic phospholipids in a calcium-dependent manner.     

Cholinergic Vesicle Specific Proteoglycan: Stability in Isolated Vesicles and in Synaptosomes During Induced Transmitter Release

Exposure of synaptosomes isolated from the electric organ of Torpedo marmorata to conditions that promote the release of acetylcholine does not cause the co-release of a vesicle specific proteoglycan. Proteoglycan within synaptosomes is quite stable during various incubation conditions as measured by immune dot blotting. Isolated vesicles from Torpedo also retain their proteoglycan immunoreactivity when exposed to a variety of incubation conditions. Lysis of vesicles in H2O, treatment with pH 11.5 buffer, or exposure to high ionic strength (2 M KCl) results in the loss of acetylcholine or ATP while the proteoglycan is retained by vesicle membranes. Only treatment with Nonidet P-40 releases proteoglycan from vesicles or synaptosomes and free proteoglycan immunoreactivity is then susceptible to degradation by trypsin or heparinase. These results suggest that the proteoglycan is an integral component of vesicle membranes and is at least in the synaptosomal preparation not subject to extensive co-release with acetylcholine or ATP.     

Phospholipid scramblase activation pathways in lymphocytes.

In erythrocytes and platelets, activation of a nonspecific lipid flipsite termed the scramblase allows rapid, bidirectional transbilayer movement of all types of phospholipids. When applied to lymphoid cells, scramblase assays reveal a similar activity, with scrambling rates intermediate between those seen in platelets and erythrocytes. Scrambling activity initiated in lymphoid cells by elevation of intracellular Ca(2+) proceeds after a lag not noted in platelets or erythrocytes. The rates of transbilayer movement of phosphatidylserine and phosphatidylcholine analogues are similar whether the scramblase is activated by elevated internal Ca(2+) or by apoptosis. Elevation of internal Ca(2+) levels in apoptotic cells does not result in an additive increase in the rate of lipid movement. In lymphoid cells from a patient with Scott syndrome, scramblase cannot be activated by Ca(2+), but is induced normally during apoptosis. These findings suggest that Ca(2+) and apoptosis operate through different pathways to activate the same scramblase.     

Capacitative and non-capacitative signaling complexes in human platelets

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.     

The separation and characterization of two forms of Torpedo electric organ calelectrin

Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ of Torpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7-8 mg.kg-1 wet weight of tissue, that is 4-5 times greater than the original method. The calelectrin so obtain could be resoloved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% beta-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32,000) that the H-form (34,000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34,000 and 32,000. The addition of 2 mM Ca2+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3-5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3nm, corresponding to an apparent Mr of 44,000. It was unaffected by changes in ionic strength, pH or Ca2+ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent Mr of 36,000. Measurements of circular dichroism indicated that 78% of the molecule was in the alpha-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.     

TRPC6 contributes to the Ca(2+) leak of human erythrocytes.

Human erythrocytes express cation channels which contribute to the background leak of Ca(2+), Na(+) and K(+). Excessive activation of these channels upon energy depletion, osmotic shock, Cl(-) depletion, or oxidative stress triggers suicidal death of erythrocytes (eryptosis), characterized by cell-shrinkage and exposure of phosphatidylserine at the cell surface. Eryptotic cells are supposed to be cleared from circulating blood. The present study aimed to identify the cation channels. RT-PCR revealed mRNA encoding the non-selective cation channel TRPC6 in erythroid progenitor cells. Western blotting indicated expression of TRPC6 protein in erythrocytes from man and wildtype mice but not from TRPC6(-/-) mice. According to flow-cytometry, Ca(2+) entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca(2+) indicator Fluo3/AM was inhibited by the reducing agent dithiothreitol and the erythrocyte cation channel blockers ethylisopropylamiloride and amiloride. Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca(2+) entry. Moreover, free Ca(2+) concentration, cell-shrinkage, and phospholipid scrambling were significantly lower in Cl(-)-depleted TRPC6(-/-) erythrocytes than in wildtype mouse erythrocytes. In conclusion, human and mouse erythrocytes express TRPC6 cation channels which participate in cation leak and Ca(2+)-induced suicidal death.     

Covalent labeling of the acetylcholine receptor from Torpedo electric tissue with the channel blocker [3H]triphenylmethylphosphonium by ultraviolet irradiation

The lipophilic cation [3H]triphenylmethylphosphonium, frequently used as a voltage sensor in membrane systems, binds reversibly to a site different from the acetylcholine binding site. This is concluded from the different pH dependences of the binding of these two ligands. Furthermore [3H]triphenylmethylphosphonium, previously identified as a channel blocker, can be covalently incorporated into acetylcholine receptor-rich membranes from Torpedo electric tissue by UV irradiation of the receptor-ligand complex. In the absence of effector, predominantly the alpha-polypeptide chains (Mr 40000) of the receptor protein are labeled by the radioactive ligand. The agonist carbamoylcholine strongly stimulates the labeling, but it directs the label predominantly to the delta- and beta-polypeptide chains. The antagonist D-tubocurarine and the virtually irreversible competitive antagonist alpha-bungarotoxin have qualitatively the same effect as the agonist carbamoylcholine. Significant differences were obtained with receptor-rich membranes prepared from Torpedo marmorata and Torpedo californica: No agonist- or antagonist-stimulated reaction was observed with the latter. The results are interpreted as an indication of a rearrangement of the receptor's quaternary structure caused by cholinergic effector binding preceding discrimination between agonists and antagonists.     

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.     

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Localization of azidophencyclidine-binding site on the nicotinic acetylcholine receptor alpha-subunit

Nicotinic acetylcholine receptors in receptor-rich membranes from Torpedo californica and from T. marmorata electric tissue were photolabeled with the non-competitive inhibitor [3H]azidophencyclidine. The receptor subunits were separated on SDS-polyacrylamide gels and the alpha-subunits recovered from the gel, were subjected to Staphylococcus aureus V8 protease cleavage. The proteolytic fragments were resolved by SDS-polyacrylamide gel electrophoresis and were identified on protein blots by 125I-labeled alpha-bungarotoxin binding and by staining with concanavalin A. The site of specific azidophencyclidine labeling has been localized to the V8-18 kDa fragment which binds toxin. Labeling of the V8-18 kDa fragment was observed in the absence and in the presence of carbamylcholine. This was found for both the species of Torpedo used here.     

ATP utilizing reactions of human erythrocyte membranes and the influence of modulator proteins

The ATP production of human erythrocytes in the steady state (approximately 2 mmoles . 1 cells-1 . h-1, 37 degrees C, pHi 7.2) is maintained by glycolysis and the ATP consumption is essentially limited to the cell membrane. About 25% of the ATP consumption is used for ion transport ATPases. The bulk of the ATP consuming processes in intact erythrocytes remains poorly understood. "Isotonic" erythrocyte membranes prepared under approximate intracellular conditions after freeze-thaw hemolysis have high (Ca2+, Mg2+)-ATPase activities (80% of the total membrane ATPase activity). There is a great discrepancy between the high capacity of the (Ca2+, Mg2+)-ATPase in isotonic membranes and the actual activity in the intact cell. The (Ca2+, Mg2+)-ATPase of isotonic membranes has a "high" Ca2+-affinity (Ka less than 0.5 microM) and a "low" Mg-ATP affinity (Km approximately 760 microM). This state of (Ca2+, Mg2+)-ATPase is caused by the association of calmodulin and 30000 Dalton polypeptides (ATP affinity modulator protein). Hypotonic washings of isotonic membranes result in a loss of the 30 kD polypeptides. EGTA (0.5 mM) extracts derived from isotonic membranes contain the 30 kD modulator protein and restore the properties of the (Ca2+, Mg2+)-ATPase of hypotonic membrane preparations to the isotonic characteristics. The Mg-ATP affinity modulator protein is assumed to form a complex with calmodulin and (Ca2+, Mg2+)-ATPase.