Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Synexin induces chromaffin granule ghosts to fuse one to another, a process which is followed continuously and quantitatively by monitoring the mixing of the intragranular aqueous compartments. A freeze-thaw technique was used for preparing chromaffin granule ghosts loaded with a self-quenching concentration of the fluorescent, high molecular weight probe FITC-Dextran. When the loaded ghosts were mixed with empty ghosts in the presence of synexin, the two compartments fused, resulting in the dilution of the probe with the concomitant increase in fluorescence. So as to suppress possible leakage signals, anti-fluorescein antibodies which quench probe fluorescence were present in the reaction media. Synexin-mediated fusion of freeze-thaw (F/Th) ghosts and binding of 125I-synexin to these membranes were found to be dependent on Ca2+ concentration, but only in a partial manner. However, these two synexin-mediated properties were demonstrably sensitive to [H+] in the medium. A detailed pH profile of fusion revealed an apparent midpoint of activation at approx. pH 5.2, with asymptotic values at pH 4 (maximum) and pH 7.2 (minimum). In our attempt to determine whether the pH effect was on the synexin or on the membranes, we found that fusion was blocked only by treatment of the membranes with the membrane-impermeant carboxyl group modifier 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. These data suggest that membrane fusion evoked by synexin seems to be promoted by rendering the F/Th membranes relatively less negatively charged while the synexin becomes more positively charged. The fusion process was entirely dependent upon synexin concentration; the k1/2 under optimal conditions of pCa and pH was 85 nM. Similar to what has been previously found with intact granules, an anti-synexin polyclonal antibody partially (48%) blocked fusion, as did pretreatment of the chromaffin granules ghosts with trypsin (30%). We conclude that the coincident pCa and pH sensitivity of synexin-mediated binding to chromaffin granule membranes and their subsequent fusion might be associated with physiological changes in the concentration of both cations in the cytoplasm of secreting chromaffin cells.
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PMID:Synexin-mediated fusion of bovine chromaffin granule ghosts. Effect of pH. 296 Mar 80

Synexin was isolated from bovine liver by high resolution cation exchange chromatography and fragmented with cyanogen bromide or trypsin. Peptides were isolated and their amino acid sequences partially determined. Twenty percent of the synexin sequence was determined in one contiguous sequence of 61 residues and a nonoverlapping sequence of 20 residues. The sequence is characterized by a hexapeptide repeat of the form YPXXXX occurring eight times in series, with phenylalanine substituting for tyrosine in two positions. The intervening amino acids (X) are predominantly proline, glycine and alanine. This pattern of periodic aromatic residues suggests the presence of a novel secondary structure and is similar to repeats present in synaptophysin, gliadin and type II keratin.
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PMID:Pattern of repeating aromatic residues in synexin. Similarity to the cytoplasmic domain of synaptophysin. 296 99

Synexin, a protein from the cytosol of the adrenal medulla, selectively increases the ability of Ca2+ to aggregate chromaffin granules and other membrane-bound particles. The ability of synexin to self-aggregate in the presence of Ca2+ can be employed in the purification of the protein by monitoring purification with parallel assays that utilize the aggregation of both chromaffin granule membranes and phosphatidylserine liposomes. It is shown that the enhancement of the Ca2+-induced aggregation of both liposomes and chromaffin granule membranes is a property associated with a 47,000 Mr protein. Trypsin inactivated synexin. We found that if granule membranes were well washed after trypsin treatment, they were still excellent substrates for synexin aggregation. This finding cannot be explained by extinction changes owing to synexin self-aggregation. The 47,000 Mr protein enhancement Ca2+ aggregation of phosphatidylserine liposomes containing up to 40% phosphatidylcholine, liposomes made from lipids extracted from chromaffin granule membranes, and trypsin-treated chromaffin granule membranes, thus suggesting that synexin activity in vivo may be independent of specific membrane proteins but dependent on the presence of acidic phospholipids in the membrane.
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PMID:Purification and mode of action of synexin: a protein enhancing calcium-induced membrane aggregation. 621 22

Interaction of clathrin coat protein with dioleoyl-phosphatidylcholine (DOPC) vesicles at pH 6.5 and below results in the formation of stable vesicle-clathrin complexes (Steer, C. J., Klausner, R. D., and Blumenthal, R. (1982) J. Biol. Chem. 257, 8533-8540). In this report we show by gel chromatography and sedimentation analysis that the interaction of clathrin coat protein with unilamellar dioleoyl phosphatidylcholine vesicles at pH = 6.0 results in the formation of larger structures. As shown by electron microscopy and an increase in trapped volume of both sucrose and inulin those larger structures represent fused bilayers. We examined the mixing of membrane lipid as a result of membrane fusion using resonance energy transfer between two fluorescent lipid probes incorporated into the same vesicle membrane. At a protein:lipid ratio of 1:500 there was 50% vesicle-vesicle fusion, at pH 6.0, as indicated by the change in efficiency of energy transfer between the fluorescent probes. Fusion was completed within 60 s. A number of other proteins (ovalbumin, rabbit IgG, trypsin, pronase, calmodulin, tubulin, synexin, bovine serum albumin) at 10-fold or higher concentrations, did not induce fusion of dioleoyl phosphatidylcholine vesicles, either at pH 7.4 or at pH 6.0. This system provides a model for pH-dependent and protein-mediated fusion of uncharged lipid bilayers.
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PMID:Clathrin-induced pH-dependent fusion of phosphatidylcholine vesicles. 682 67

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.
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PMID:Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7. 1590 72