Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.
Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.
Hayashi
H
H
Department of Oncogene Research, Osaka University, Japan.
Owada
M K
MK
Sonobe
S
S
Domae
K
K
Yamanouchi
T
T
Kakunaga
T
T
Kitajima
Y
Y
Yaoita
H
H
D017302:Annexins; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D010740:Phospholipases / Q000037:antagonists & inhibitors*; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012867:Skin / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D017302:Annexins; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium