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Calmodulin Affinity for Brain Coated Vesicle Proteins
Author(s) -
Moskowitz Nathan,
Schook William,
Lisanti Michael,
Hua Elaine,
Puszkin Saul
Publication year - 1982
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1982.tb06657.x
Subject(s) - calmodulin , vesicle , chemistry , synaptic vesicle , biophysics , biochemistry , microbiology and biotechnology , biology , enzyme , membrane
A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of [ 125 I‐labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin‐C. Scatchard analysis revealed one high‐affinity, low‐capacity binding site, K D = 3.9 ± 0.6 n M , B max = 16.3 ± 2.4 pmol/mg, and one low‐affinity, high‐capacity binding site, K D = 102 ± 15.0 n M , B max = 151 ± 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 μg calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X‐100‐treated extract of coated vesicles was passed through a Sepharose 4B‐calmodulin affinity column, polypeptides with M r s (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca 2+ ‐dependent manner. A 30,000 M r , protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca 2+ ‐Mg 2+ ]‐ATPase activity as well as Ca 2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 M r calmodulin binding proteins represent the [Ca +2 ‐Mg 2+ ]‐ATPase complex, the other coated vesicle calmodulin binding site.