RESUMEN
Phospholipase C (Bacillus cereus) contains two apparently essential and very reactive lysine residues that may be labelled selectively by pyridoxal 5'-phosphate [Aurebekk & Little (1977) Biochem, J. 161, 159--165]. One of these lysine residues was found in the 25-amino acid N-terminal fragment liberated by CNBr digestion of the pyridoxal-labelled enzyme and identified as lysine-6. Two of the labelled peptides isolated from the chymotryptic digest of pyridoxal-labelled enzyme contained proline, suggesting that the other labelled lysine residue is situated in the same region of the primary structure as the single proline residue of the enzyme.
Asunto(s)
Bacillus cereus/enzimología , Lisina/análisis , Fosfolipasas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Aminoácidos/análisis , Sitios de Unión , Cromatografía en Gel , Quimotripsina/metabolismo , Fragmentos de Péptidos/análisis , Fosfato de PiridoxalRESUMEN
A very simple and rather unusual purification scheme for phospholipase C from Bacillus cereus has been worked out. Air is bubbled vigorously through the bacterial culture and the foam collected. Liquified foam is centrifuged, dialyzed and heated at 74 degrees C for 5 min. After centrifugation, affinity chromatography is carried out on lipoprotein-Sepharose. The enzyme is then thermally denatured by exposure to 85 degrees C for 5 min and the precipitated material well washed and then renatured from solution in 4 M guanidinium chloride. The final enzyme preparations are electrophoretically homogeneous and easy to crystallize. The recovery of activity exceeds 80%.