title: Improved purification and enzymatic properties of a mixture of Sticholysin I and II: isotoxins with hemolytic and phospholipase A(2) activities from the sea anemone Stichodactyla helianthus.
abstract: Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10(-3)U/mg of protein). In this work, a mixture StI-StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI-StII was relatively high (1.85U/mg) and dependent of Ca(2+). The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40mM Ca(2+) at pH 8.0. This Ca(2+) concentration did not exert any effect on binding of StI-StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: adelmonte@fbio.uh.cu.
authors: Alberto
authors: A
authors: del Monte-Martínez
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: jogoba@fbio.uh.cu.
authors: Jorge
authors: J
authors: González-Bacerio
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: laz@fbio.uh.cu.
authors: Lázara
authors: L
authors: Romero
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: eduardo@bioplantas.cu.
authors: Carlos
authors: C
authors: Aragón
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: dmcb@fbio.uh.cu.
authors: Diana
authors: D
authors: Martínez
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: mchavez@infomed.sld.cu.
authors: María
authors: M
authors: de Los Á Chávez
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: calvarez@fbio.uh.cu.
authors: Carlos
authors: C
authors: Álvarez
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: mlanio@fbio.uh.cu.
authors: María E
authors: ME
authors: Lanio
authors: Instituto de Catálisis, CSIC, Campus UAM, Canto Blanco, 28049 Madrid, España. Electronic address: jmguisan@icp.csic.es.
authors: José M
authors: JM
authors: Guisán
authors: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, 25 # 455 entre J e I, Cuba. Electronic address: jcdiaz@infomed.sld.cu.
authors: Joaquín
authors: J
authors: Díaz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D003064:Cnidarian Venoms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012615:Sea Anemones / Q000737:chemistry* / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D003064:Cnidarian Venoms; D006460:Hemolysin Proteins; D009930:Organic Chemicals; C404794:sticholysin II; C121348:stycholysin I; D054467:Phospholipases A2; D002118:Calcium
keywords: Affinity chromatography; Hemolysin; PLA(2); Stichodactyla helianthus; Sticholysin I; Sticholysin II