Purification, biological properties and partial sequence analysis of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard.
Calcimedin is a group of proteins, originally isolated from chicken gizzard, which are able to bind to several hydrophobic matrices in the presence of Ca2+. Although the molecular properties have been partially discovered, the physiological functions of calcimedins have not yet been clearly defined. In this study, we describe the isolation and characterization of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard. Both structural and functional studies establish that 67-kDa calcimedin is a member of the calpactin/lipocortin family: it displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding and phospholipid binding activity similar to those of calpactins (lipocortins). By comparing the sequence of 67-kDa calcimedin with the predicted sequence of 67-kDa calelectrin, we concluded that the primary structure of these 67-kDa proteins is highly conserved. In particular, the sequences GLGTDEGAIIXVLTQR and EGAGTDESTLIEIMATR conform with the annexin consensus sequence which is characteristic of the calpactin/lipocortin family. A 34-kDa fragment of 67-kDa calcimedin was also purified and their relatedness has been confirmed by antibody cross-reactivity. The sequence data further support that the 34-kDa fragment is derived from the C-terminal portion of 67-kDa calcimedin by limited proteolysis. The 34-kDa fragment, which contains the annexin consensus sequence, preserves the phospholipase A2 inhibitory activity, and binds F-actin and phospholipids.
Kobayashi
R
R
Department of Biochemistry, Akita University School of Medicine, Japan.
Tashima
Y
Y
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017302:Annexins; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D002645:Chickens; D003429:Cross Reactions; D005895:Gizzard, Avian / Q000032:analysis*; D006801:Humans; D007124:Immunoenzyme Techniques; D008081:Liposomes / Q000032:analysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D010743:Phospholipids / Q000032:analysis; D013329:Structure-Activity Relationship; D014357:Trypsin
D000199:Actins; D000596:Amino Acids; D017302:Annexins; D002135:Calcium-Binding Proteins; D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin; D002118:Calcium