GEL-IMMOBILIZED PROTEIN PHOSPHATASE 2A FROM YARROWIA-LIPOLYTICA DEPHOSPHORYLATES PHOSVITIN AND MODIFIES ITS FUNCTIONAL-PROPERTIES

The catalytic subunit of a protein phosphatase 2A (PP2Ac) purified fro m Yarrowia lipolytica was immobilized by covalent coupling on CNBr-Sep harose 4B with a fixation yield of about 70-85%. The specific activity of free PP2Ac for substrate phosvitin was almost totally preserved by the immobilization process. The immobilized enzyme exhibits strongly improved thermostability. Phosvitin dephosphorylation by immobilized P P2Ac attained 14% and 35% yield after 3 and 17 h incubation, respectiv ely, and resulted in modified phosvitin properties, e.g., improved sol ubility and alteration of ultraviolet absorption spectrum. Electrophor etic data indicated that beta-phosvitin was preferentially dephosphory lated by the immobilized enzyme. Presence of a reductant such as DTT i n the reaction medium improved dephosphorylation efficiency by inducin g formation of a phosphate complex which would prevent enzyme inhibiti on by the released phosphate. This work indicates that immobilization of protein phosphatases is a performant tool to achieve modifications of highly phosphorylated proteins through dephosphorylation, with easy retrieval of the modified protein from the reaction medium.