title: Characterizations of two distinct Ca2+-dependent phospholipid-binding proteins of 68-kDa isolated from human placenta.
abstract: Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.
authors.affiliation: Department of Oncogene Research, Osaka University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
authors.forename: M K
authors.initials: MK
authors.lastname: Owada
authors.forename: S
authors.initials: S
authors.lastname: Sonobe
authors.forename: T
authors.initials: T
authors.lastname: Kakunaga
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017317:Annexin A6; D017302:Annexins; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution
chemical_list: D000199:Actins; D017317:Annexin A6; D017302:Annexins; D002135:Calcium-Binding Proteins; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium