Identification of annexin II, annexin VI and glyceraldehyde-3-phosphate dehydrogenase as calcyclin-binding proteins in bovine heart.
1. Matrix-immobilized calcyclin as affinity ligand in chromatography led to purification of three protein bands at 68, 36 and 35 kDa from bovine heart that required Ca2+ for binding. 2. Polyacrylamide-immobilized phosphatidylserine separated this fraction into a phospholipid-binding part (68 kDa, 35 kDa), also attaching to phospholipid vesicles even in the presence of calcyclin, and a flow-through part, constituting approx 30% of the total fraction (36 kDa). 3. Enzyme assays and electrophoretic mobility showed an at least close relationship of the 36 kDa band to glyceraldehyde-3-phosphate dehydrogenase. Interaction between enzyme and calcyclin in a solid-phase assay was inhibited by sialoglycoproteins and depended strongly on the integrity of carboxyl and hydrophobic groups of the enzyme. The interaction between the two proteins had a KD value of 110 nM. 4. Application of annexin-specific antibodies revealed an immunological relationship of the 35 and 68 kDa calcyclin-binding proteins to members of the annexin family, namely to annexin II (35 kDa) and annexin VI (68 kDa). The N-terminal amino acid sequence of a cleavage peptide of the 68 kDa protein was identical to a sequence stretch in human annexin VI, corroborating this evidence.
Zeng
F Y
FY
Institut für Pharmazeutische Chemie, Marburg, Germany.
Gerke
V
V
Gabius
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000302:isolation & purification / Q000378:metabolism*; D017317:Annexin A6 / Q000302:isolation & purification / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D009418:S100 Proteins
D017306:Annexin A2; D017317:Annexin A6; D002135:Calcium-Binding Proteins; D010743:Phospholipids; D009418:S100 Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases