Advertisement

Parasitology Research

, Volume 84, Issue 4, pp 310–314 | Cite as

Isolation of an Entamoeba histolytica intracellular alkaline phospholipase A2

  • J. Vargas-Villarreal
  • A. Olvera-Rodríguez
  • B. D. Mata-Cárdenas
  • H. G. Martínez-Rodríguez
  • S. Said-Fernández
  • A. Alagón-Cano
ORIGINAL PAPER

Abstract

The major hemolytic activity of Entamoeba histolytica is located in a subcellular fraction called P30. Its maximal effect is observed at pH 8.0 and 1 mM Ca2+ and is due to a phospholipase A (PLA). In the present study a membrane-associated phospholipase A2 was purified from P30 to homogeneity. P30 was fractionated with ethyl ether and the insoluble fraction was extracted with 1 M KCl. The KCl-soluble material was diluted ten times with 0.1 M TRIS-HCl (pH 9.5) and passed through a chromatofocusing column with a 9–4 pH gradient. Four peaks with PLA2 activity were obtained. By affinity chromatography, peak II, the one with the highest specific activity, was resolved in three more PLA2 peaks. Peak II.2 had the highest PLA2 specific activity. When analyzed by sodium dodecyl sulfate-polyacrylamide slab-gel electrophoresis under nonreducing conditions, peak II.2 yielded a single band with an apparent molecular mass of 30 kDa. Under reducing conditions the protein dissociated into two 15-kDa monomers. The purified PLA II.2 displayed its activity at the same conditions under which the P30 hemolytic activity was maximal. The isoelectric point of PLA II.2 was 7.0. The purification procedure described above provides sufficient material for determination of the relative importance of the enzyme in the E. histolytica pathogenic mechanisms.

Keywords

Ethyl Ether Purification Procedure Subcellular Fraction Hemolytic Activity High Specific Activity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • J. Vargas-Villarreal
    • 1
  • A. Olvera-Rodríguez
    • 1
  • B. D. Mata-Cárdenas
    • 2
  • H. G. Martínez-Rodríguez
    • 3
  • S. Said-Fernández
    • 2
  • A. Alagón-Cano
    • 1
  1. 1.Departamento de Reconocimiento Molecular y Bioestructura, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, N.L. MexicoMX
  2. 2.División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, N.L. MexicoMX
  3. 3.Unidad de Laboratorios de Ingeniería y Expresión Genética, Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, N.L. MexicoMX

Personalised recommendations