Calcium-dependent binding of the plasma protein apolipoprotein A-I to two members of the annexin family.
Affinity chromatography with purified annexins coupled to CNBr-activated Sepharose 4B was used to determine the capacity of proteins found in cytosolic fractions of the bovine adrenal medulla to bind to an immobilized annexin in a Ca2+-dependent manner. Several proteins were eluted from a recombinant annexin I column in the presence of 2 mM EGTA, including protein kinase C (PKC), members of the annexin family, and a 26 kDa protein that appeared as the most prominent band on SDS-PAGE. The identities of PKC, annexin I, annexin IV, annexin VI, and annexin VII were confirmed by Western blotting. The 26 kDa protein was purified by anion exchange chromatography on a Poros Q column and determined to be apolipoprotein A-I (apoA-I) by peptide sequencing. Comigration of apoA-I and chromobindin 2 on two-dimensional gels identified apoA-I as chromobindin 2. Overlay assays were performed to verify the apoA-I-annexin I interaction using apoA-I immobilized on nitrocellulose and annexin I in solution with binding detected using anti-annexin I antiserum. Additionally, the ability of biotin-labeled apoA-I in solution to bind to several purified annexins immobilized on nitrocellulose was determined by detection with horseradish peroxidase-conjugated avidin. Using these methods, it was shown that both annexin I and annexin VII bind to bovine apoA-I in a Ca2+-dependent manner. Other annexins, such as annexin IV and annexin VI, do not exhibit this binding. The results suggest that certain annexins may function as extracellular binding sites for plasma proteins.
Brownawell
A M
AM
Department of Pharmacology, University of Virginia, Charlottesville 22908, USA. amb7c@Virginia.EDU
Creutz
C E
CE
D000313:Adrenal Medulla / Q000378:metabolism; D000818:Animals; D017305:Annexin A1 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017314:Annexin A4 / Q000378:metabolism; D017317:Annexin A6 / Q000378:metabolism; D017310:Annexin A7 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016632:Apolipoprotein A-I / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D017305:Annexin A1; D017314:Annexin A4; D017317:Annexin A6; D017310:Annexin A7; D016632:Apolipoprotein A-I; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011493:Protein Kinase C; D002118:Calcium