A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family

• Published in: The Journal of biological chemistry Vol. 267; no. 13; pp. 8919 - 8924
• Main Authors: TOKUMITSU, H, MIZUTANI, A, MINAMI, H, KOBAYASHI, R, HIDAKA, H
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.05.1992
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Annexins; Autoradiography; Binding and carrier proteins; Biological and medical sciences; Calcium - metabolism; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - isolation & purification; Calcium-Binding Proteins - metabolism; Cations, Divalent; Chromatography, Affinity; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Lung - chemistry; Molecular Sequence Data; Multigene Family; Phospholipids - metabolism; Proteins; Rabbits; S100 Proteins; Sequence Homology, Nucleic Acid; Binding protein; Ligand binding; Vertebrata; Mammalia; Purification; Affinity chromatography; Rabbit; Lipocortin; Phospholipid; Lagomorpha
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)50367-2

A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.