VAT-1 from Torpedo synaptic vesicles is a calcium binding protein: a study in bacterial expression systems.
1. Calcium binding properties were examined in VAT-1, an abundant 41-kDa membrane protein expressed in the cholinergic cynaptic vesicles of Torpedo. 2. An overlay assay, using 45Ca2+ as a tracer, demonstrated the ability of a recombinant VAT-1 produced from the IPTG-inducible pKK223-3 expression vector to bind calcium. 3. A high yield of recombinant VAT-1 was obtained from the glutathione S-transferase (GST) expression system. The fusion product enabled VAT-1 purification via affinity chromatography. Subsequent cleavage by thrombin resulted in its separation from the GST carrier protein. 4. A direct Ca(2+)-binding study was performed with purified VAT-1 by a quick-spin column technique, in the presence of 45Ca2+. Quantitative analysis revealed a 1:1 molar stoichiometry for binding of Ca2+ to VAT-1, with a dissociation constant of 130 microM. 5. A GST-linked truncated protein consisting of 13 kDa from the VAT-1 carboxy-terminal domain was found to retain the capacity to bind Ca2+. 6. A data search for homologies between VAT-1 and known Ca(2+)-binding proteins revealed considerable similarity to members of the annexin family in a 140-amino acid region from the carboxy terminal of VAT-1, which overlaps two tandem Ca(2+)-binding domains of the annexin proteins.
Levius
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O
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Linial
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M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013572:Synaptic Vesicles / Q000378:metabolism*; D014101:Torpedo
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C063277:VAT-1 protein, Torpedo; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D002118:Calcium