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An affinity column for phospholipase A2 based on immobilised acylaminophospholipid analogues.
Dijkman, R; Beiboer, S H; Verheij, H M.
Afiliación
  • Dijkman R; Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
Biochim Biophys Acta ; 1347(1): 1-8, 1997 Jul 12.
Article en En | MEDLINE | ID: mdl-9233682
ABSTRACT
A synthetic route was developed to prepare 2-acylamino phospholipid analogues suitable for immobilisation. The inhibitors, synthesised in either the (R)- and (S)-configuration, carried an omega-carboxyl group in one acyl chain for immobilisation to the matrix. As a matrix Sepharose 6B, derivatised with a polar, non-charged 16 atom spacer was used. Low-molecular weight phospholipase A2 binds in a calcium-dependent way to the immobilised (S)-inhibitor and not to the immobilised (R)-inhibitor which shows that binding involves specific active site interactions rather than hydrophobic chromatography. The specificity was further demonstrated by the fact that the immobilised (S)-inhibitor binds porcine pancreatic and snake venom phospholipases A2, but not the porcine pancreatic zymogen. Moreover, a mutant porcine pancreatic phospholipase A2 in which the active side residue His48 has been replaced by Gln, was not bound by the column. This column material might be applicable for affinity purification of phospholipase A2 and for screening of phage display libraries.
Asunto(s)
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Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Fosfolipasas A / Cromatografía de Afinidad Límite: Animals Idioma: En Revista: Biochim Biophys Acta Año: 1997 Tipo del documento: Article País de afiliación: Países Bajos
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Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Fosfolipasas A / Cromatografía de Afinidad Límite: Animals Idioma: En Revista: Biochim Biophys Acta Año: 1997 Tipo del documento: Article País de afiliación: Países Bajos